Detection

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What are some initial indicators for the presence of a HAB?

Initial detection of freshwater HAB events relies on qualitative, visual observations. The tell-tale manifestations of a HAB include:
  • surface water discoloration (e.g., a red, green, or brown tint)
  • thick, mat-like accumulations on the shoreline and surface
  • fish kills.

What should you consider when collecting samples of water containing cyanobacteria/cyanotoxins?

Samples should reflect the water source conditions and handled properly to ensure reliable results. 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 that have been exposed to any treatment chemicals should be quenched immediately upon sampling. Sodium thiosulfate or ascorbic acid are commonly used as quenching agents.
  • 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 (taking precautions to avoid breakage) may be appropriate to extend holding times.

When to do cell lysing?

It is important to isolate a pure culture of the cyanobacterial strain and characterize and quantify the toxin to confirm the cyanobacterial strain producing the toxin. When measuring “total” cyanotoxins (both intracellular and dissolved (extracellular) toxins), rupturing cyanobacterial cells (lysing) is generally employed to break the cell wall and release the toxins into solution. Freeze/thaw cycling (traditionally carried out over three or more cycles) represents the most common lysing technique, though some analytical methods rely on other approaches. Lysing is particularly important for samples collected prior to the PWS filter effluent. For a well-designed, well-operated PWS lysing would not be expected to have a significant impact on finished water (post-filtration) samples as cyanobacteria cells should not be present at significant levels in the finished water. Some analysts elect to confirm the effectiveness of raw-water lysing (or to judge the need for finished-water lysing) using microscopic examination for intact algal cells.

What detection methods are available for cyanobacteria and cyanotoxins in water?

There is a diverse range of rapid screen tests and laboratory methods used 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.

Analysis of microcystins is most commonly carried out using reversed-phase high performance liquid chromatographic methods (HPLC) combined with mass spectrometric (MS, MS/MS) or ultraviolet/photodiode array detectors (UV/PDA). Analytical methods such as enzyme–linked immunosorbent assays (ELISA) already exist to analyze cyanobacterial hepatotoxins and saxitoxins, and the protein phosphatase inhibition assay (PPIA) can be used for microcystins. These two methods are sensitive, rapid, and suitable for large-scale screening but are predisposed to false positives and unable to differentiate between toxin variants. The liquid chromatography/mass spectrometry (LC/MS) method can be fast in identifying the toxicants in the samples. Conventional polymerase chain reaction (PCR), quantitative real–time PCR (qPCR) and microarrays/DNA chips can be used to detect microcystin/nodularin and saxitoxin producers. 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.

For detection of cyanotoxins in drinking water, EPA developed Method 544, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for microcystins and nodularin (combined intracellular and extracellular), and Method 545, a LC-ESI/MS/MS method for the determination of cylindrospermopsin and anatoxin-a.  EPA also developed Method 546, an Adda ELISA Method.

The following table describes the methods available for cyanotoxin measurement in freshwater. For more information, please review Analytical Methods for Cyanotoxin Detection and Impacts on Data Interpretation by Keith Loftin, Jennifer Graham, Barry Rosen (U.S. Geological Survey) and Ann St. Amand (Phycotech). The presentation was given on April 26, 2010 at the 2010 National Water Quality Monitoring Conference at Denver, CO.

Key:
✓: Available for this toxin
0: Unavailable for this toxin

A Summary of the Methods Available for Cyanotoxin Detection *

* The following table describes the methods available for the most common cyanotoxins in freshwater.

Method Freshwater Cyanotoxins Description *
 ANA-a  CYL   MC 
 Biological Assays (Class Specific Methods at Best)
Mouse  ✓  ✓  ✓ Qualitative screening assay. (Detection limit: NA). 
Protein Phosphate Inhibition Assays (PPIA)  ✓  0  ✓ Useful as a screening tool; relatively simple to use and is highly sensitive, with low detection limits relative to any current guideline values. Currently, there is no commercial kit available. (Detection imit: Around 0.1 µg L-1 or less)1
Neurochemical  ✓  0  0 Qualitative screening assay. Helpful for determining whether or not toxins are present. (Detection limit: NA).
Enzyme-Linked Immunosorbent Assays (ELISA)  ✓  ✓  ✓ Semi-quantitative screening assay capable of detecting low toxin concentrations. (Detection limit: 0.05 µg L-1)1.
 Chromatographic Methods (Compound Specific Methods)
 Gas Chromatography
Gas Chromatography with Flame Ionization (GC/FID)  ✓  0  0 Uses a Hydrogen/Air flame into which the sample is passed to oxidize organic molecules, which produces ions. These ions are collected and produce an electrical signal which is then measured. Derivitization is typically required. (Detection limit: found to be as low as 0.004 µg L-1; however, detection limit depends on the water concentration of the sample)1.
Gas Chromoatography with Mass Spectrometry (GC/MS)  ✓  0  0 Separates the components of the sample and then characterizes each component qualitatively and quantitatively. Derivitization is typically required. (Detection limit: 0.011 µg L-1)2
 Liquid Chromatography
Liquid Chromatography/Utraviolet-Visible Detection (LC/UV or HPLC)  ✓  ✓  ✓ Distinct from traditional liquid chromatography because operational pressures are significantly higher. Variable specificity; subject to interference with co-eluting matrix. (Detection limit: 1.0 µg L-1)3.
Liquid Chromatography/Flourescence (LC/FL)  ✓  0  0 Variable specificity; subject to interference with co-eluting matrix. Usually requires post column oxidation prior to detection. (Detection limit: 0.025 µg L-1)4
 Liquid Chromatography combined with mass spectrometry (high specificity)
Liquid Chromatography Ion Trap Mass Spectrometry (LC/IT MS)  ✓  ✓  ✓ Second in compound specificity only to LC/TOF MS. (Detection limit: Reported as low as 0.002 µg L-1; however, varies based upon cyanotoxin class and sampling procedure)5.
Liquid Chromatography Time-of-Flight Mass ( LC/TOF MS)  ✓  ✓  ✓ Accurate mass capability makes this technique most specific of LC-MS techniques. (Detection limit: Reported as low as 0.001 µg L-1)6.
Liquid Chromatography Single Quadrupole Mass Spectrometry (LC/MS)  ✓  ✓  ✓ Weaker specificity than LC/MS/MS. Slow scanning speed relative to other mass analyzers. (Detection limit: 0.5 µg L-1)7.
Liquid Chromatography Triple Quadrupole Mass Spectrometry (LC/MS/MS)  ✓  ✓  ✓ Routinely employed, third most specific of LC-MS techniques. The number of compounds that can be simultaneously analyzed in a single run is limited. (Detection limit: Ranges from 0.8 µg L-1 to 3.2 µg L-1)8.

Acronyms:

ANA-a: Anatoxin-a
CYL: Cylindrospermopsin
MC: Microcystin

References:

1 CRC for Water Quality and treatment – Research Report 74.
2 Koreiviene, J. and O. Belous (2012). “Methods for Cyanotoxins Detection.” Botanica Lithuanica, Vol 18, pp 58-65.
3 Ghassempour, A. et al. (2005). “Analysis of anatoxin-a using polyaniline as a sorbent in solid-phase microextraction coupled to gas chromatography-mass spectrometry.” Journal of Chromatography, Vol 1078, pp 120-127.
4 Fawell, J.K., H.A. James (1994). “Toxins from Blue-Green Algae: Toxicological Assessment of Anatoxin-a and a Method for its Determination in Reservoir Water.” Report No. FR0434, Foundation for Water Research.
5 Osswald, J., et al. (2009). “Production of anatoxin-a by cyanobacterial strains isolated from Portuguese freshwater systems.” Ecotoxicology, Vol 18, pp. 1110-1115.
6 Perez, S. and D. S. Aga (2005). “Recent advances in the sample preparation, liquid chromatography tandem mass spectrometric analysis and environmental fate of microcystins in water.” TrAC Trends in Analytical Chemistry, Vol 24, pp 658-670.
7 Sancho, A., et al. (2006). “Potential of liquid chromatography/time-of-flight mass spectrometry for the determination of pesticides and transformation products in water.” Analytical and Bioanalytical Chemistry, Col 386, pp987-997.
8 Al-Sammak, M. A., et al (2013). “Methods for simultaneous detection of the cyanotoxins BMAA, DABA, and anatoxin-a in environmental samples.”Toxicon, Vol. 76, pp 216-325

Who has the capability to perform these detection methods?

In the case of public waterways and drinking water sources, many state environmental agencies operate monitoring, sampling, and testing programs. Several of these states perform the necessary detection analysis on samples taken from potential HABs in state-run laboratories; however, many states with HAB programs, in addition to municipalities, private utilities, and other riparian stakeholders of freshwater systems send their samples to commercial and public laboratories. For a non-comprehensive list of laboratories that accept samples for cyanobacteria and cyanotoxin analysis, please visit the State Resources page on this website.

More Information

US EPA Method 545. Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water by Liquid Chromatography Electrospray ionization Tandem Mass Spectrometry (LC/ESI-MS/MS)
US EPA Method 544. Determination of Microcystins and Nodularin in Drinking Water by Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)
US EPA Video: Laboratory Techniques for Detecting Microcystins in Water Using Enzyme-Linked Immunosorbent Assay (ELISA)
US EPA Presentations EPA Workshop on Cyanobacteria and Cyanotoxins Occurrence and Detection Methods, July 2012
Cyanobacterial Toxin Cylindrospermopsin
US EPA Drinking Water Health Advisory for the Cyanobacterial Toxin Cylindrospermopsin
US EPA Drinking Water Health Advisory for the Cyanobacterial Microcystins Toxins
US EPA Environmental Technology Verification Program, Immunoassay Test for Microcystins
USGS Field and laboratory guide to freshwater cyanobacteria harmful algal blooms for Native American and Alaska Native Communities (PDF) (54 pp, 8 MB, About PDF)
USGS Guidelines for Design and Sampling for Cyanobacterial Toxin and Taste-and-Odor Studies in Lakes and Reservoirs
USGS Analytical Methods for Cyanotoxin Detection and Impacts on Data Interpretation MERHAB-LGL - Analytical Techniques webpage
Microcystins ELISA Test Kits Health Canada Algal Toxin Tests Kits Report
Indiana Department of Environmental Management, Blue-Green Algae Sampling Resource List (PDF) (2 pp, 184 K, About PDF) Exit

For comments, feedback or additional information, please contact Lesley D'Anglada (Danglada.Lesley@epa.gov), Project Manager, at 202-566-1125.

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