ASTM D3978-21a

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Designation: D3978 − 21 D3978 − 21a
Standard Practice for
Algal Growth Potential Testing with Pseudokirchneriella
1,2
subcapitata
This standard is issued under the fixed designation D3978; 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 (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Algae are natural inhabitants of surface waters and are found in almost every water environment
that is exposed to sunlight. The algae contribute to purification (both organic and inorganic) of streams
and lakes and are necessary as food for fish and fish food organisms. When large amounts of nutrients
are available, excessive growths referred to as “blooms” can occur. Some algal blooms release
substances toxic to fish, birds, domestic animals, and other alga. When nutrients are exhausted, the
growth of algae and production of oxygen by photosynthesis decreases. The respiration of bacteria
decomposing the large quantity of algal cells can deplete dissolved oxygen to the extent that fish and
other oxygen consumers die. Both the abundance and composition of algae are related to water quality,
with algal growth primarily influenced by the availability of nutrients.
The presence of indigenous algae in a water sample suggests that they are the most fit to survive
in the environment from which the sample was taken. The indigenous algae should produce biomass
until limited from further growth by some essential nutrient. If the indigenous algal production is
limited from further growth by an essential nutrient, the laboratory test alga cultured in a
noncompetitive environment and responding to the same limiting nutrient will produce parallel
maximum yield growth responses. Generally, indigenous phytoplankton bioassays are not necessary
unless there is strong evidence of the presence of long-term sublethal toxicants to which indigenous
populations might have developed tolerance (1) .
A single-indigenous algal species, dominant at the time of sampling, may not be more indicative of
natural conditions than a laboratory species that is not indigenous to the system. The dynamics of
natural phytoplankton blooms, in which the dominant algal species changes throughout the growth
season, makes it quite certain that even if the indigenous algal isolate was dominant at the time of
collection, many other species will dominate the standing crop as the season progresses.
When comparing algal growth potentials from a number of widely different water sources there are
advantages in using a single species of alga. The alga to be used must be readily available and its
growth measured easily and accurately. It must also respond to growth substances uniformly. Because
some algae are capable of concentrating certain nutrients in excess of their present need when they are
grown in media with surplus nutrients, this factor must be taken into account in selecting the culture
media and in determining the type and amount of algae to use. (2) showed that a blue-green algae
Microcystis aeruginosa, cultured in a low-nitrogen concentration medium, would not grow when
transferred to medium lacking nitrogen. However, when the alga was cultured in medium containing
four times as much nitrogen it was able to increase growth two-fold after transfer into nitrogen-free
This practice is under the jurisdiction of ASTM Committee E50 on Environmental Assessment, Risk Management and Corrective Action and is the direct responsibility
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Jan. 15, 2021Nov. 1, 2021. Published February 2021January 2022. Originally approved in 1980. Last previous edition approved 20122021 as
D3978-04 (Reapproved 2012). DOI: 10.1520/D3978-21.-21. DOI: 10.1520/D3978-21A.
Renamed by Gunnar Nygaard, Jirf Komárek, Jørgen Kristiansen and Olav M. Skulberg, 1986. Taxonomic designations of the bioassay alga NIVA-CHL1 ("Selenastrum
capricornutum") and some related strains. Opera Botanica 90:5-46.
The boldface numbers in parentheses refer to the references at the end of this practice.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D3978 − 21a
medium. A green alga Pseudokirchnereilla subcapitata (also known as Selenastrum capricornutum
and Raphidocelis subcapitata), gave a similar response. In an analogous experiment with phosphorus,
both organisms increased four-fold when transferred to medium lacking phosphorus. However, if
algae are cultured in relatively dilute medium as recommended in the Algal Assay Procedure: Bottle
Test (3) for culturing Pseudokirchnereilla subcapitata, disclosed no significant further growth in
medium lacking nitrogen or phosphorus when these were transferred from the initial medium over a
wide range of inoculum sizes (4).
There are several methods available for determining algal growth. Measurements of optical density,
oxygen production, carbon dioxide uptake, microscopical cell counts, and gravimetric cell mass
determinations have been used, but often lack sensitivity when the number of cells is low.
Measurement of the uptake of carbon-14 in the form of bicarbonate is a sensitive method but can also
be time-consuming. In vivo fluorescence of algal chlorophyll has been used with many types of algae
and has proved particularly useful with indigenous algae or filamentous forms not easily measured at
low concentrations by other methods. The method is sensitive and measurements can be quickly
performed. However, chlorophyll to cell mass ratio may vary significantly with growth in water
samples of different chemical composition (5). The electronic particle counter has been used for
counting and sizing nonfilamentous unialgal species (6,7). Shiroyama, Miller, and Greene (8) have
developed a procedure for using an electronic particle counter to count and size Anabaena flos-aquae
filaments cultured in natural waters.
The need for standardization of techniques for measuring the potential for algal growth was
recognized by the Joint Industry/Government Task Force on Eutrophication (9). Thereafter, the
Environmental Protection Agency developed, in association with industrial and university
cooperation, a Bottle Test for assaying algal growth potential in natural water samples (3). An
expanded and improved version of the Bottle Test was published in 1978 (10). It is this work on which
the following test is based.
1. Scope Scope*
1.1 This practice measures, by Pseudokirchnereilla subcapitata growth response, the biological availability of nutrients, as
contrasted with chemical analysis of the components of the sample. This practice is useful for assessing the impact of nutrients,
and changes in their loading, upon freshwater algal productivity. Other laboratory or indigenous algae can be used with this
practice. However, Pseudokirchnereilla subcapitata must be cultured as a reference alga along with the alternative algal species.
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use. For a specific precautionary statement, see Section 1516.
1.3 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
E729 Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
E943 Terminology Relating to Biological Effects and Environmental Fate
E1023 Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses
E1733 Guide for Use of Lighting in Laboratory Testing
SI10–16 IEEE/ASTM SI-10 American National Standard for Metric Practice
3. Terminology
3.1 Definitions:
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
D3978 − 21a
3.1.1 The words “must”, “should”, “may”, “can”, and “might” have very specific meanings in this guide. “Must” is used to express
an absolute requirement, that is, to state that the test ought to be designed to satisfy the specified condition, unless the purpose of
the test requires a different design. “Must” is used only in connection with factors that directly relate to required test procedures
(see Section 14). “Should” is used to state that the specified condition is recommended and ought to be met if possible. Although
violation of one “should” is rarely a serious matter, violation of several will often render the results questionable. Terms such as
“is desirable,” “is often desirable,” and “might be desirable” are used in connection with less important factors. “May” is used to
mean “is (are) allowed to,” “can” is used to mean “is (are) able to,” and “might” is used to mean “could possibly.” Thus, the classic
distinction between “may” and “can” is preserved, and “might” is never used as a synonym for either “may” or “can.”
3.1.2 For definitions of other terms used in this guide, refer to Terminologies D1129 and E943 and Guide E729. For an
explanation of units and symbols, refer to SI10–16.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 spike, v—to add a material or compound to the test matrix (for example, water) for experimental purposes.
4. Summary of Practice
4.1 A water sample is filtered or autoclaved and filtered, placed in a covered Erlenmeyer flask, inoculated with the test algal
species, and incubated under constant temperature and light intensity until the increase in biomass is less than 5 % per day
(generally between day 7 and 14). Nutrients may also be added to aliquots of the sample to determine growth controlling nutrients.
5. Significance and Use
5.1 The significance of measuring algal growth potential in water samples is that differentiation can be made between the nutrients
of a sample determined by chemical analysis and the nutrients that are actually available for algal growth. The addition of nutrients
(usually nitrogen and phosphorus singly or in combination) to the sample can give an indication of which nutrient(s) is (are)
limiting for algal growth (1,10,11,12,13,14).
6. Interferences
6.1 Autoclaving may cause precipitation of certain constituents in the sample and elevate the pH. These precipitates are not
necessarily irreversible or unavailable as nutrients. The sample may often be clarified by equilibrating it in a CO atmosphere
followed by equilibration in air to its original pH.
6.2 Toxic substances in the sample may affect the growth response of the algae.
7. Apparatus
7.1 Water Sampler, nonmetallic.
7.2 Sample Container—Linear polyethylene bottles.
7.3 Centrifuge, capable of 1000 g.
7.4 Environmental Chamber, with temperature control (24 6 2 °C) and illumination (cool white fluorescent or LED lights) that
provides 4300 lm/m 610 % (Lumens/square meter aka lux), or equivalent (400 640 footcandle) (see Guide E1733).
7.5 Shaker, rotary, capable of 100 to 120 rpm.
7.6 Flasks, Erlenmeyer, 250-mL.
NOTE 1—Other sizes are acceptable as long as the liquid does not exceed 50 % of the total flask volume.
D3978 − 21a
7.7 Flask Covers, Beakers, or Foam Plugs—Some foam plugs, upon autoclaving, may release substances toxic to the test algae.
Each laboratory, when changing its source of supply, must determine whether the new closures have a significant effect on the
maximum standing crop.
7.8 Tubes, graduated centrifuge.
7.9 Pipets, Eppendorf or equivalent, with disposable tips, 0.1 or 1.0 mL.
7.10 Filtration Apparatus, nonmetallic, with vacuum or pressure source.
7.11 Membrane Filters, sterile 0.22-μm or 0.45-μm particle size retention, low-water extractable.
7.12 Balance, analytical, capable of weighing 100 g with a precision of 60.1 mg.
7.13 Autoclave.
7.14 pH Meter.
7.15 Calibrated Light Meter, reading in μ , lumens or footcandles.
mol m–2s–1
7.16 Particle Counter and Mean Cell Volume Accessory, with 100-μm aperture.
7.17 Compound Microscope, capable of 100×.
7.18 Hemocytometer.
7.19 Fluorometer, equipped to measure chlorophyll a, or
7.20 Spectrophotometer, to measure cell densities in log phase cultures.
8. Reagents
8.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the committee on Analytical Reagents of the American Chemical Society. Other
grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity to permit its use without lessening
the accuracy of the determination.
8.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water conforming to
Specification D1193, Type III.
8.3 Calcium Chloride Solution—Dissolve 1.66 g of CaCl in 500 mL of water.
8.4 Magnesium Chloride Solution—Dissolve 6.08 g of MgCl ·6H O in 500 mL of water.
2 2
8.5 Magnesium Sulfate Solution—Dissolve 3.59 g of MgSO in 500 mL of water.
8.6 Micro Nutrient Solutions (Note 2)—Dissolve the following in 500 mL of water:
“Reagent Chemicals, American Chemical Society Specifications,” Am. Chemical Soc., Washington, DC. For suggestions on the testing of reagents not listed by the
American Chemical Society, see “Reagent Chemicals and Standards,” by Joseph Rosin, D. Van Nostrand Co., Inc., New York, NY, and the “United States Pharmacopeia.”
D3978 − 21a
NOTE 2—Reagents 7.38.3, 7.48.4, 7.68.6, and 7.98.9 can be combined into one stock solution.
93 mg of boric acid (H BO )
3 3
208 mg of manganous chloride (MnCl ·4H O)
2 2
1.6 mg of zinc chloride (ZnCl )
80 mg of ferric chloride (FeCl ·6H O)
3 2
0.39 mg of cobalt chloride (CoCl )
3.63 mg of sodium molybdate (NaMoO ·2H O)
4 2
0.006 mg of cupric chloride (CuCl ·2H O)
2 2
150 mg of ethylenediaminetetraacetic acid
(HOCOCH ) N(CH ) H(HOCOCH )
2 2 2 2 2 2
8.7 Potassium Phosphate Solution—Dissolve 0.52 g of K HPO in 500 mL of water.
2 4
8.8 Sodium Bicarbonate Solution—Dissolve 7.50 g of NaHCO in 500 mL of water.
8.9 Sodium Nitrate Solution—Dissolve 12.75 g of NaNO in 500 mL of water.
9. Preparation of Culture Flasks
9.1 Brush the inside of flasks with a stiff bristle brush to loosen any attached materials.
9.2
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