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Designation: D 3731 – 87 (Reapproved 1998)

Standard Practices for Measurement of

Chlorophyll Content of Algae in Surface Waters1
This standard is issued under the fixed designation D 3731; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.

1. Scope 4. Significance and Use

1.1 These practices include the extraction and the measure- 4.1 Data on the chlorophyll content of the algae have the
ment of chlorophyll a, b, and c, and pheophytin a in freshwater following applications:
and marine plankton and periphyton. Three practices are 4.1.1 To provide estimates of algal biomass and productiv-
provided as follows: ity.
1.1.1 Spectrophotometric, trichromatic practice for measur- 4.1.2 To provide general information on the taxonomic
ing chlorophyll a, b, and c. composition (major groups) of the algae, based on the relative
1.1.2 Spectrophotometric, monochromatic practice for mea- amounts of chlorophyll a, b, and c, and the physiological
suring chlorophyll a corrected for pheophytin a; and for condition of algal communities, which is related to the relative
measuring pheophytin a. abundance of pheopigments.
1.1.3 Fluorometric practice for measuring chlorophyll a 4.1.3 To determine long-term trends in water quality.
corrected for pheophytin a; and for measuring pheophytin a. 4.1.4 To determine the trophic status of surface waters.
1.2 The values stated in SI units are to be regarded as the 4.1.5 To detect adverse effects of pollutants on plankton and
standard. The values given in parentheses are provided for periphyton in receiving waters.
information purposes only. 4.1.6 To determine maximum growth rates and yields in
1.3 This standard does not purport to address all of the algal growth potential tests.
safety problems, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- 5. Interferences and Special Considerations
priate safety and health practices and determine the applica- 5.1 Pigment Extraction—The chlorophylls are only poorly
bility of regulatory limitations prior to use. Specific precau- extracted, if at all, from some forms of algae, such as the
tionary statements are given in Section 7. coccoid green algae, unless the cells are disrupted, whereas
other algae, such as the diatoms, give up their pigments very
2. Terminology readily when merely steeped in acetone. Since natural commu-
2.1 Definitions: nities of algae usually consist of a wide variety of taxa that
2.1.1 plankton—nonmotile or weakly swimming organisms, differ in their resistance to extraction, it is necessary to disrupt
usually microscopic, that drift or are carried along by currents the cells routinely to ensure maximum recovery of the chloro-
in surface waters, commonly consisting of bacteria, algae, phylls. Failure to do so may result in a systematic underesti-
protozoa, rotifers, and microcrustacea. mation of 10 % or more in the chlorophyll content of the
2.1.2 periphyton—microorganisms growing on submerged samples. (1, 2, 3)2
objects, commonly consisting of bacteria, algae, protozoa, and 5.2 Grinders—The cells of many common coccoid green
rotifers. algae resist disruption by most methods, but usually yield their
pigments after maceration with a tissue grinder. The routine
3. Summary of Practices use of grinders, therefore, is recommended. Glass-to-glass
3.1 The chlorophyll and related compounds are extracted grinders are more rigorous in disrupting cells in plankton
from the algae with 90 % aqueous acetone. The concentration concentrated by centrifugation, and in periphyton scrapings,
of the pigments is determined by measuring the light absorp- than are TFE-fluorocarbon-to-glass grinders, and their use for
tion or fluorescence of the extract. this purpose is preferred. However, TFE fluorocarbon-to-glass
grinders perform well with glass-fiber filters. Other cell dis-
1
ruption methods, such as sonication, may be used if, for each
These practices are under the jurisdiction of ASTM Committee E-47 on
Biological Effects and Environmental Fate and are the direct responsibility of
Subcommittee E47.01 on Aquatic Assessment and Toxicology.
2
Current edition approved Sept. 25, 1987. Published November 1987. Originally The boldface numbers in parentheses refer to the list of references at the end of
published as D 3731-79. Last previous edition D 3731-79. this standard.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.

1
 D 3731
type of sample, it is demonstrated that the chlorophyll recovery 6. Apparatus
is comparable to that obtained with tissue grinders (4). 6.1 Filters, Glass-fiber filters, providing quantitative reten-
5.3 Filters—Glass-fiber filters usually provide a higher tion of particles equal to or greater than 0.45 µm in diameter.
recovery of chlorophyll than is obtained with membrane filters 6.2 Filtering Apparatus suitable for use with glass-fiber
when extraction-resistant algae are present in the samples, and filters.
should be employed routinely (4). 6.3 Tissue Homogenizer—Tissue grinder consisting of a
5.4 Chlorophyll-Related Pigments—Naturally occurring, motor-driven pestle and enclosing glass tube (glass to glass or
structurally related chlorophyll precursors and degradation TFE-fluorocarbon-to-glass grinder).4
products, such as the chlorophyllides, pheophytins, and 6.4 Spectrophotometer suitable for use over the range from
pheophorbides, commonly occur in pigment extracts and may 600 to 750 nm, with a resolution of 2 nm or better, and
absorb light in the same region of the spectrum as the equipped with sample cells having a light path of 1, 5, and 10
chlorophylls. These compounds may interfere with the analysis cm, with a capacity of 10 mL or less.
by indicating falsely high chlorophyll concentrations. 6.5 Fluorometer (Optional):
5.4.1 This practice includes a correction for pheophytin a 6.5.1 Spectrophotofluorometer that provides an excitation
only. Pheophytin a is similar in structure to chlorophyll a, but wavelength of 430 nm and detection of emission over the range
lacks the magnesium atom (Mg) in the porphyrin ring. The from 600 to 700 nm, or:
magnesium can be removed from chlorophyll in the laboratory 6.5.2 Filter Fluorometer equipped with a blue light source
by acidifying the extract. When a solution of pure chlorophyll and blue excitation filter5 and a sharp cut off filter3
a is converted to pheophytin a by acidification, the absorption 6.6 Centrifuge that can provide a centrifugal force of 1000
peak is reduced to approximately 60 % of its original value and g; head with swing-out buckets preferred.
shifts from 664 to 665 nm, resulting in a before:after acidifi- 6.7 Centrifuge Tubes, screw-cap or stoppered, conical,
cation absorption peak ratio (OD664/OD665) of 1.70. This graduated, 15-mL. Avoid cap liners soluble in acetone and
phenomenon is utilized in correcting the apparent concentra- neoprene rubber stoppers.
tion of chlorophyll a for the presence of pheophytin a.
Unwanted degradation of chlorophyll to pheophytin in the 7. Reagents and Materials
phytoplankton on filters, or in periphyton samples, or in the 7.1 Aqueous Acetone, 90 %—Add 1 volume of distilled
acetone extract, by the occurrence of acidic conditions can be water to 9 volumes of reagent grade acetone. Add 5 drops of 1
prevented by the addition of a magnesium carbonate suspen- N sodium bicarbonate solution per litre.
sion to the plankton sample before filtering or to the periphyton
samples before grinding, and by adding a small amount of a NOTE 1—Caution: the volume:volume relationship between the ac-
etone and water must be strictly followed to prevent shifts in the
sodium bicarbonate solution to the aqueous acetone when it is absorption peaks.
prepared. Addition of magnesium carbonate may also aid in
clarifying the samples following steeping (5). 7.2 Hydrochloric Acid (1 N)—Add 1 volume of concen-
5.5 Turbidity—The optical density of the extract is mea- trated hydrochloric acid (HCl, sp gr 1.19) to eleven volumes of
sured at 750 nm to correct for turbidity. distilled water.
5.6 Spectrophotometer Resolution—The absorption peak of 7.3 Magnesium Carbonate Suspension—Add 1 g of finely
acetone solutions of chlorophyll extracts is relatively narrow, powdered magnesium carbonate to 100 mL of distilled water in
and a spectrophotometer with a resolution of 2 nm or better is a stoppered Erlenmeyer flask. Shake immediately before use.
required to obtain accurate results. If instruments of lower 7.4 Sodium Bicarbonate Solution (1 N)—Prepare by dis-
resolution are employed, the concentration of chlorophyll a solving 8.4 g of sodium bicarbonate in 100 mL of distilled
may be significantly underestimated depending on the band water.
width. At a spectral band width of 20 nm, the error in the
8. Sampling
estimate of the chlorophyll a concentration may be as large as
40 %. 8.1 Plankton:
5.7 Fluorometer Filters—In the fluorometric practice, inter- 8.1.1 Collection—Collect samples with a water bottle, dia-
ferences from light emitted by chlorophyll b and chlorophyll c phragm pump, or other suitable device. To protect the chloro-
are greatly reduced by the use of a sharp cut-off red filter3 that phyll from degradation prior to extraction and analysis, imme-
blocks all light with a wavelength of less than 650 nm (6). diately add 1 mL of magnesium carbonate suspension per L of
5.8 Light Sensitivity of Extracts—Chlorophyll solutions de- sample, and protect from the direct sunlight.
grade rapidly in strong light. Work with these solutions, 8.1.2 Concentration—Immediately concentrate the plank-
therefore, should be carried out in subdued light, and all ton by filtering or by centrifuging for 20 min at 1000 g. To
vessels, tubes, etc., containing the pigment extracts should be avoid cell damage and loss of contents during filtration, do not
covered with aluminum foil or other opaque substance.

4
Kontes type C, glass-to-glass grinder or its equivalent, has been found suitable
for this purpose. Available from Kontes Manufacturing Co., Spruce St., Vineland,
3
Corning CS-2-64 filter or its equivalent, has been found suitable for this NJ 08360.
5
purpose. Available from Corning Glass Works, 388 Beartown Rd., Painted Post, NY Corning CS-5-60 filter has been found satisfactory. Equivalent filters may be
14870. used.

2
 D 3731
exceed a vacuum of 1⁄2 atm (50 kPa). After centrifuging, check 10.2.1 The chlorophyll concentration is a function of the
the samples for buoyant cells that may resist sedimentation. absolute optical density (OD) of the extract at the specified
8.1.3 Holding Time—Samples that cannot be concentrated wavelengths, rather than the relative OD as is commonly the
immediately after collection may be held at 0 to 4°C in the dark case in colorimetric analyses. For quantitative chlorophyll
for 24 h before the plankton are concentrated. The centrifugate determinations, it is essential, therefore, to check the instru-
or residue on the filter may be stored in the dark at −20°C for ment or chart OD readings, or both, at several wavelengths in
30 days before extracting the pigment. the range from 600 to 750 nm across the full span of the
8.2 Periphyton: absorbance (OD = 0.0–1.0), using a set of filters6 of known
8.2.1 Collection—Take samples from natural or artificial optical density.
substrates and immediately ice, freeze, or place in cold (iced) 10.2.2 Transfer the extract to a sample cell and measure the
aqueous acetone in the dark. optical density at 750, 664, 647, and 630 nm. If possible,
8.2.2 Holding Time—Iced samples may be held 24 h before choose a cell-path length or dilution to provide an OD664
they are further processed. Frozen samples may be held greater than 0.2 and less than 1.0.
at −20°C for 30 days. 10.2.3 Subtract the OD750 from each of the other ODs.
Then divide by the cell-path length in centimetres.
9. Pigment Extraction
10.2.4 Calculate the concentration of chlorophyll a, b, and c
9.1 Algal cells in plankton concentrates (from centrifuga- in the extract by inserting the 1-cm OD664, OD647, and
tion or filtration) and periphyton scrapings are disrupted by OD630 into the following Jeffrey and Humphrey equations (6):
grinding in a tissue homogenizer for 3 min at approximately
Chl a, mg/L 5 11.85~OD664! 2 1.54~OD647! 2 0.08~OD630!
500 r/min in 4 to 5 mL of 90 % aqueous acetone. Use a
(1)
glass-to-glass tissue grinder for macerating plankton concen-
trate obtained by centrifugation and for macerating periphyton Chl b, mg/L 5 21.03~OD647! 2 5.43~OD664! 2 2.66~OD630!
(2)
scrapings. A TFE-fluorocarbon-to-glass or glass-to-glass
grinder may be used to macerate plankton concentrated on Chl c, mg/L 5 24.52~OD630! 2 1.67~OD664! 2 7.60~OD647!
glass-fiber filters. (3)
9.2 Wash the homogenate into a vial or into a 15-mL 10.2.5 Express the concentration of pigments in a plankton
centrifuge tube, rinse the pestle and grinding tube with a small sample as milligrams per cubic metre (mg/m3) and calculate as
amount of aqueous acetone, bring the volume of the extract to follows:
10 mL by adding 90 % aqueous acetone, cap or stopper the Ca 3 E
tube, and mix and place the material in the dark at 4°C to steep. Chl a, mg/m 3 5 G (4)
9.3 Steep not less than 15 min or more than 24 h. Mix the
homogenate by inverting the tube several times, and clarify the where:
extract by centrifuging 20 min at 1000 g, or by filtering. If the Ca = concentration of chlorophyll a in the extract, mg/L,
clarified extract is not analyzed immediately, store in the dark E = extract volumes, L, and
at −20°C in a tightly stoppered tube. G = grab sample volume, m3.
9.4 After clarification, decant the extract directly into a 10.2.6 Express the concentration of pigments in a periphy-
cuvette or a screw-cap or stoppered tube. If the analysis can not ton sample as milligrams per square metre (mg/m2) and
be carried out immediately, the extract can be stored for 1 year calculate as follows:
without appreciable chlorophyll degradation if held in the dark Ca 3 E
at −20°C. Chl a, mg/m 2 5 A (5)

10. Extract Analysis where:

10.1 The two practices of extract analysis commonly em- Ca = concentration of chlorophyll a in the extract, mg/L,
ployed are visible spectrophotometry and fluorometry (6, 7). and
Each practice has its advantages and disadvantages. The A = substrate area sampled, m2.
trichromatic, spectrophotometric practice, has the advantage of 10.3 Spectrophotometric, Monochromatic Practice:
providing a simple procedure for the simultaneous estimation 10.3.1 Measure the optical density of the chlorophyll extract
of chlorophyll a, b, and c, which, at the current state of at 750 and 664 nm.
technology, cannot be obtained as easily by the fluorometric 10.3.2 Acidify the extract by adding 2 drops of 1 N HCl
practice, but does not correct for chlorophyll degradation (amount added to a 1-cm cell; if a larger cell is used, add a
products. The monochromatic, spectrophotometric practice proportionately larger volume of acid), stir well, and measure
corrects the chlorophyll a for pheophytin, a, but does not the OD at 750 and 665 nm not sooner than 1 min or later than
measure chlorophyll b and c. The fluorometric practice, is one 2 min after acidification.
to three orders of magnitude more sensitive than the spectro-
photometric practices, when using the instrumentation com-
monly employed for chlorophyll analyses, but the practices for 6
Filters, Standard Reference Material 930, Glass Filters for Spectrophotometry,
simultaneously measuring chlorophyll b and c are much more available from the National Bureau of Standards, Office of Product Standards,
complex. Administration Building A603, Gaithersburg, MD 20899, or equivalent have been
10.2 Spectrophotometric, Trichromatic Practice: found suitable for this purpose.

3
 D 3731
10.3.3 Calculate the concentration of chlorophyll a (Ca) where:
corrected for the presence of pheophytin a, and the concentra- Ca = concentration of chlorophyll a, µg/L, and
tion of pheopigments expressed as pheophytin a (Pa) in the R = fluorometer reading.
extract by inserting the 1-cm ODs in the following equations 10.4.3 To correct for the presence of pheopigments, ex-
(5): pressed as pheophytin a, determine a before:after acidification
Ca, mg/L 5 26.7~OD664b 2 OD665a! (6) fluorescence ratio, r, as described in 10.3.2 using an extract that
is free of pheophytin a, where the before:after acidification
Pa, mg/L 5 26.7@1.7~OD665a! 2 OD664b# (7) ratio is 1.70, based on the OD664b/OD665a, as determined
with a spectrophotometer. Use the fluorometric before:after
where: acidification ratio (r) and the calculation factor, F (s), in the
OD664b = OD664 − OD750 measured before acidifica- following equations:
tion, and r
OD665a = OD665 − OD750 measured after acidification. Ca, µg/L 5 F~s! r 2 1 ~Rb 2 Ra! (9)
10.3.4 Calculate the concentration of these pigments in
plankton and periphyton samples as described in 10.2.5 and
10.2.6, respectively. r
Pa, µg/L 5 F~s! r 2 1 ~rRa 2 Rb! (10)
10.4 Fluorometric Practice:
10.4.1 The fluorometric practice is 10 to 1000 times more
sensitive than the spectrophotometric practices and requires where:
Ca and Pa = concentration of chlorophyll a and pheophy-
proportionately smaller amounts of sample. The method has
tin a, respectively, in the extract,
important disadvantages, however, which include the inability
Rb = fluorometer reading before acidification; and
to easily determine chlorophyll b and c concentrations, and the Ra = fluorometer reading after acidification.
need to calibrate the instrument with “reference” chlorophyll
solutions containing a known concentration of chlorophyll a
11. Precision and Bias
determined by previous spectrophotometric analysis (7).
10.4.2 To calibrate the instrument, carefully dilute the 11.1 It is not practicable to specify the precision of the
“reference” extract to provide solutions that give midscale procedures in Practices D 3731 for measuring chlorophyll
readings in each sensitivity range of the fluorometer. Use the content of algae since there are no interlaboratory data sets
readings to determine a calibration factor, F (s), for each available at this time.
sensitivity level (s) as follows:
Ca
12. Keywords
F~s! 5 R (8) 12.1 algae surface water; chlorophyll; pheophytin

REFERENCES

(1) Golterman, H. L., “Methods for Chemical Analysis of Fresh Waters,” Limnology and Oceanography, Vol 16, No. 6, 1971, pp. 990–992.
International Biological Program Handbook No. 8. Blackwell Scien- (5) Lorenzen, C. J., “Determination of Chlorophyll and Phaeopigments:
tific Publication, London, 1969, p. 172. Spectrophotometric Equations,” Journal of Limnology and Oceanog-
(2) Strickland, J. D. H., and Parsons, T. R., “A Practical Handbook of raphy, Vol 12, pp. 343–346.
Seawater Analysis,” Bulletin Fisheries of the Research Board of (6) Jeffrey, S. W., and Humphrey G. F., “New Spectrophotometric
Canada, No. 167, Ottawa, 1968, p. 311. Equations for Determining Chlorophylls a, b, c1 and c2 in Higher
(3) Vollenweider, R. A., ed., “A Manual on Methods for Measuring Plants, Algae, and Natural Phytoplankton,” Biochemistry and Physi-
Primary Production in Aquatic Environments,” International Biologi- ology of Plants, Vol 167, ZEB Gustav Fischer Verlag, East Germany,
cal Program Handbook No. 12, Second Edition, Blackwell Scientific 1975, pp. 191–194.
Publication, London, 1974, p. 225. (7) Holm-Hansen, O., Lorenzen, C. J., Holmes, L. W., and Strickland J. D.
(4) Long, E. V., and Cooke, G. D., “A Quantitative Comparison of H., “Fluorometric Determination of Chlorophyll,” Journal of Cons.
Pigment Extraction by Membrane and Glass-Fiber Filters,” Journal of Int. Explor. Mer. Vol 30, No. 1, pp. 3–15.

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