NEMI Method Summary

Method Summary Information

Method Number: 446.0 Media: WATER
Revision: Revision 1.2, September 1997
Method Source: U.S. EPA National Exposure Research Laboratory (NERL) [formerly EMSL]
Analytes in this method
Subcategory: BIOCHEMICAL
Official Name: In Vitro Determination of Chlorophylls a, b, c1 + c2 and Pheopigments in Marine and Freshwater Algae by Visible Spectrophotometry
Descriptive Name Chlorophylls and Pheopigments in Phytoplankton by Spectrophotometry
Source Info: U.S.EPA National Exposure Research Laboratory (NERL)
Microbiological and Chemical Exposure Assessment Research Division (MCEARD)
[formerly the Environmental Monitoring Systems Laboratory (EMSL), Cincinnati, OH]
26 West Martin Luther King Drive
Cincinnati, Ohio 45268-0001
Fax: 513-569-7757
email: DWmethods.help@epa.gov
Citation: Methods for Determination of Chemical Substances in Marine and Estuarine Matrices - 2nd Edition (EPA/600/R-97/072)
175914 Byte file
Brief Method
Summary:
 Chlorophyll-containing phytoplankton in a measured volume of sample water are concentrated by filtration at low vacuum through a glass fiber filter. The pigments are extracted from the phytoplankton in 90% acetone with the aid of a mechanical tissue grinder and are allowed to steep 2-24 hours. The resulting slurry is centrifuged to clarify the solution, and the absorbance of the supernatant liquid is measured at 4 wavelengths to determine turbidity, and chlorophylls a, b, and c1 + c2. Pheopigment-correct chl a can be determined by using absorbance measurements from an acidified and non-acidified sample. Absorbance values are entered into a set of equations to that utilize the extinction coefficients of the pure pigments in 90% acetone to simultaneously calculate the concentrations of the pigments in a mixed solution.
Scope And
Application:
 This method determines chlorophylls a, b and c1 + c2, and pheopigments of chlorophyll a found in marine and freshwater phytoplankton.
Applicable
Conc Range:
Interferences: (A) Absorbing materials, generally: Any material that absorbs near the measured wavelengths can interfere with measurements. High absorption at 750 nm (>= 0.005 AU) indicates a poorly clarified solution, requiring centrifugation or filtration prior to analysis.

(B) Spectral overlap of pigments: The relative amounts of chlorophyll a, b, and c1 + c2 vary with the taxonomic composition of the phytoplankton. Due to the spectral overlap of the chlorophylls and pheopigments, over- or underestimation of the pigments is inevitable in solutions containing all of these pigments. Knowledge of taxonomic composition of samples and proper analytical technique can reduce errors, as discussed extensively in the method.

(C) Steeping: The amount of sample steeping can effect precision and recovery by effecting extraction of target pigments and of interfering substances.

(D) Temperature and light: All photosynthetic pigments are light and temperature sensitive. Work must be performed in subdued light and all standards, QC materials, and filtered samples must be stored in the dark at -20 or -70oC to prevent rapid degradation.
QC Requirements: Each laboratory using this method is required to implement a formal quality control (QC) program. The minimum requirements of this program consist of an initial demonstration of performance, continued analysis of laboratory reagent blanks, field replicates and QC samples as a continuing check on performance.
Sample Handling: Samples are collected using a pump or grab sampler. Enough water should be collected to concentrate phytoplankton on three or more filters. Four liters may be required for ocean water (where phytoplankton density is usually low), whereas one liter is generally sufficient for lake, bay, and estuarine water. The method provides detailed information on the procedure for collecting samples. Once collected and filtered, the sample may be stored 2-4 hours on ice in the dark, but should be stored at -20 or -70oC as soon as possible. Filters frozen at -20oC may be stored as long as 3.5 weeks without significant loss of chlorophyll a.
Max Holding Time:
 3.5 weeks at -20oC
 
Relative Cost: Unknown
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