ASTM E1366-23

Standard Practice for Standardized Aquatic Microcosms: Fresh Water

SIGNIFICANCE AND USE
5.1 A microcosm test is conducted to obtain information concerning toxicity or other effects of a test material on the interactions among three trophic levels (primary, secondary, and detrital) and the competitive interactions within each trophic level. As with most natural aquatic ecosystems, the microcosms depend upon algal production (primary production) to support the grazer trophic level (secondary production), which along with the microbial community are primarily responsible for the nutrient recycling necessary to sustain primary production. Microcosm initial condition includes some detritus (chitin and cellulose) and additional detritus is produced by the system. The microcosms include ecologically important processes and organisms representative of ponds and lakes, but are non-site specific. To the extent possible, all solutions are mixtures of distilled water and reagent grade chemicals (see Section 8) and all organisms are available in commercial culture collections.  
5.2 The species used are easy to culture in the laboratory and some are routinely used for single species toxicity tests (Guide E729; Practice D3978, Guides E1192 and E1193). Presumably acute toxicity test results with some of these species would be available prior to the decision to undertake the microcosm test. If available, single species toxicity results would aid in distinguishing between indirect and direct effects.  
5.3 These procedures are based mostly on published methods (4-6), interlaboratory testing (7-10, 11), intermediate studies (12-23, 24), statistical studies  (25-27) and mathematical simulation results  (28). Newer studies on jet fuels have been reported (29)(See 15.1 for multivariate statistical analyses) and on the implications of multispecies testing for pesticide registration (30). Environmental Protection Agency, (EPA) and Food and Drug Administration, (FDA) published similar microcosm tests (31). The methods described here were used to determine the criteria for A...
SCOPE
1.1 This practice covers procedures for obtaining data concerning toxicity and other effects of a test material to a multi-trophic level freshwater community, independent of the location of the test.  
1.2 These procedures also might be useful for studying the fate of test materials and transformation products, although modifications and additional analytical procedures might be necessary.  
1.3 Modification of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting multi-trophic level tests.  
1.4 This practice is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Practice  
4  
Significance and Use  
5  
Apparatus  
6  
Facilities  
6.1  
Container  
6.2  
Equipment  
6.3  
Hazards  
7  
Microcosm Components  
8  
Medium  
8.1  
Medium Preparation  
8.2  
Sediment  
8.3  
Microcosm Assembly  
8.4  
Test Material  
9  
General  
9.1  
Stock Solution  
9.2  
Nutrient Control  
9.3  
Test Organisms  
10  
Algae  
10.1  
Animals  
10.2  
Specificity of Organisms  
10.3  
Sources  
10.4  
Algal Culture Maintenance  
10.5  
Animal Culture Maintenance  
10.6  
Procedure  
11  
Experimental Design  
11.1  
Inoculation  
11.2  
Culling  
11.3  
Addition of Test Material  
11.4  
Measurements  
11.5  
Reinoculations  
11.6  
Analytical Methodology  
12  
Data Processing  
13  
Calculations of Variables from Measurements  
14  
Statistical Analyses ...

General Information

• Status: Published
• Publication Date: 31-May-2023
• ICS: 13.060.30 - Sewage water
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Refers: ASTM E943-08 - Standard Terminology Relating to Biological Effects and Environmental Fate
• Effective Date: 01-Mar-2008
• Refers: ASTM E1733-95(2008) - Standard Guide for Use of Lighting in Laboratory Testing
• Effective Date: 01-Feb-2008
• Refers: ASTM E1192-97(2008) - Standard Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Feb-2008
• Refers: ASTM E729-96(2007) - Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Oct-2007
• Refers: ASTM E1023-84(2007) - Standard Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses
• Effective Date: 01-Oct-2007
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D1193-99e1 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Refers: ASTM D1193-99 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999

Overview

ASTM E1366-23 - Standard Practice for Standardized Aquatic Microcosms: Fresh Water outlines procedures for evaluating the ecological effects and toxicity of various materials on artificial, multi-trophic level freshwater communities. Developed by ASTM Committee E50, this international standard enables reliable, repeatable testing of chemical and biological impacts across multiple levels of the aquatic food web, simulating the complexity of natural pond or lake ecosystems.

The method employs standardized aquatic microcosms, focusing on interactions among primary producers (algae), grazers (such as Daphnia), and detritivores, alongside nutrient cycling and competitive dynamics. The practice facilitates laboratory-based studies using well-defined, commercially available organisms and chemically defined media, supporting consistent results independent of geographic location.

Key Topics

• Multi-trophic Level Testing: The standard enables studies across three ecological levels: primary producers (algae), secondary (grazers), and detrital (decomposers).
• Chemical and Ecological Assessment: Test materials can be evaluated for acute or chronic toxicity, transformation, and ecological effects.
• Standardized Conditions: Use of commercially available organisms and distilled water with reagent-grade chemicals ensures reproducibility.
• Experimental Design: The protocol addresses setup, inoculation, culling, addition of test material, reinoculation, and comprehensive measurements (e.g., organism abundance, nutrient concentrations, oxygen, pH).
• Data and Statistical Analysis: The practice includes methods for data collection, processing, and one-way analysis of variance to compare test and control groups.

Applications

ASTM E1366-23 is widely applicable in the following areas:

• Environmental Risk Assessment: Supports product registration and regulatory compliance by providing data on how chemicals affect aquatic ecosystems, including indirect and population-level effects not captured by single-species tests.
• Toxicity Testing: Useful for determining both direct and indirect impacts of chemicals and transformation products, enhancing understanding of ecosystem-level effects.
• Chemical Fate Studies: While primarily ecological, the protocol can inform studies on the fate and transport of chemicals in freshwater systems.
• Product and Chemical Development: Helps industries assess potential environmental impacts of new compounds, including pesticides, pharmaceuticals, and industrial chemicals.
• Academic and Methodological Research: Researchers can utilize this practice to develop or validate novel test procedures and explore innovative microcosm designs.

Limitations include the absence of vertebrates, limited predation, and the non-gnotobiotic nature (not free from unknown microbes) of the microcosms, which limits direct extrapolation to all natural water bodies.

Related Standards

For broader context in aquatic toxicity and microcosm research, the following ASTM standards are frequently referenced alongside ASTM E1366-23:

• ASTM E729: Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
• ASTM D3978: Practice for Algal Growth Potential Testing with Pseudokirchneriella subcapitata
• ASTM E1192/E1193: Guides for Conducting Life-Cycle Toxicity Tests with Aquatic Organisms
• ASTM E943: Terminology Relating to Biological Effects and Environmental Fate
• ASTM E1023: Guide for Assessing Hazards of Materials to Aquatic Organisms
• EPA and FDA Microcosm Tests: Further guidelines published by regulatory agencies often align with or supplement this practice.

Practical Value

Implementing ASTM E1366-23 provides:

• Repeatable, standardized results for ecological risk assessments
• Improved insight into community-level impacts of chemical and biological agents
• Regulatory acceptance due to international recognition and harmonized procedures
• Adaptability for specific research or compliance needs by permitting certain procedural modifications (with clear documentation for comparability)

This standard supports more holistic, realistic assessments of potential aquatic environmental impacts, addressing complex interactions that single-species protocols may overlook.

Standards Content (Sample)

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.
Designation: E1366 − 23
Standard Practice for
Standardized Aquatic Microcosms: Fresh Water
This standard is issued under the fixed designation E1366; 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.
1. Scope
Algae 10.1
Animals 10.2
1.1 This practice covers procedures for obtaining data
Specificity of Organisms 10.3
concerning toxicity and other effects of a test material to a Sources 10.4
Algal Culture Maintenance 10.5
multi-trophic level freshwater community, independent of the
Animal Culture Maintenance 10.6
location of the test.
Procedure 11
Experimental Design 11.1
1.2 These procedures also might be useful for studying the
Inoculation 11.2
fate of test materials and transformation products, although
Culling 11.3
Addition of Test Material 11.4
modifications and additional analytical procedures might be
Measurements 11.5
necessary.
Reinoculations 11.6
Analytical Methodology 12
1.3 Modification of these procedures might be justified by
Data Processing 13
special needs or circumstances. Although using appropriate
Calculations of Variables from Measurements 14
procedures is more important than following prescribed Statistical Analyses 15
Acceptability of Test 16
procedures, results of tests conducted using unusual procedures
Interpretation of Results 17
are not likely to be comparable to results of many other tests.
Report 18
Annex Annex A1
Comparison of results obtained using modified and unmodified
Appendices
versions of these procedures might provide useful information
Relationship of Media Appendix X1
concerning new concepts and procedures for conducting multi-
Statistical Guidance Appendix X2
trophic level tests.
1.5 The values stated in SI units are to be regarded as the
1.4 This practice is arranged as follows: standard. The values given in parentheses are for information
only.
Section
1.6 This standard does not purport to address all of the
Referenced Documents 2
safety concerns, if any, associated with its use. It is the
Terminology 3
Summary of Practice 4
responsibility of the user of this standard to establish appro-
Significance and Use 5
priate safety, health, and environmental practices and deter-
Apparatus 6
mine the applicability of regulatory limitations prior to us-
Facilities 6.1
Container 6.2
e.Specific hazard statements are given in Section 7.
Equipment 6.3
1.7 This international standard was developed in accor-
Hazards 7
dance with internationally recognized principles on standard-
Microcosm Components 8
Medium 8.1
ization established in the Decision on Principles for the
Medium Preparation 8.2
Development of International Standards, Guides and Recom-
Sediment 8.3
Microcosm Assembly 8.4 mendations issued by the World Trade Organization Technical
Test Material 9
Barriers to Trade (TBT) Committee.
General 9.1
Stock Solution 9.2
2. Referenced Documents
Nutrient Control 9.3
Test Organisms 10
2.1 ASTM Standards:
D1193 Specification for Reagent Water
D3978 Practice for Algal Growth Potential Testing with
This practice is under the jurisdiction of ASTM Committee E50 on Environ-
mental Assessment, Risk Management and Corrective Action and is the direct
responsibility of Subcommittee E50.47 on Biological Effects and Environmental
Fate. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved June 1, 2023. Published July 2023. Originally approved contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
in 1990. Last previous edition approved in 2016 as E1366 – 11(2016). DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E1366-23. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1366 − 23
Pseudokirchneriella subcapitata 3.3.4 community metabolism, n—the oxygen or carbon di-
E729 Guide for Conducting Acute Toxicity Tests on Test oxide balance of the entire community.
Materials with Fishes, Macroinvertebrates, and Amphib-
3.3.4.1 Discussion—In this microcosm, community metabo-
ians lism is estimated by the gain in oxygen during the lighted
E943 Terminology Relating to Biological Effects and Envi-
period (an estimate of net photosynthesis—P) and the loss of
ronmental Fate oxygen during the dark period (an estimate of respiration—R).
E1023 Guide for Assessing the Hazard of a Material to
When expressed as a P/R ratio, a value of >1 indicates that
Aquatic Organisms and Their Uses autotrophic processes are dominant; a value of <1 indicates that
E1192 Guide for Conducting Acute Toxicity Tests on Aque-
heterotrophic processes are dominant. If the difference of P and
ous Ambient Samples and Effluents with Fishes, R are considered (P-R), a positive number indicates autotrophic
Macroinvertebrates, and Amphibians processes are dominant, and a negative number indicates
E1193 Guide for Conducting Daphnia magna Life-Cycle heterotrophic processes are dominant. Because P and R often
Toxicity Tests change in the same direction and magnitude, P/R maybe less
E1733 Guide for Use of Lighting in Laboratory Testing sensitive than P or R considered separately.
IEEE/SI 10 American National Standard for Use of the
3.3.5 detritivore, n—an organism that feeds on detritus,
International System of Units (SI): The Modern Metric
dead organic material.
System
3.3.6 ecosystem, n—a system made up of a community of
animals, plants, and bacteria and its interrelated physical and
3. Terminology
chemical environment (3).
3.1 The words “must,” “should,” “may,” “can,” and “might”
3.3.7 gnotobiotic, adj—a culture which the exact composi-
have very specific meanings in this practice. “Must” is used to
tion of the organisms is known, down to the presence or
express an absolute requirement, that is, to state that the test
absence of bacteria.
ought to be designed to satisfy the specific condition, unless the
3.3.7.1 Discussion—Such cultures are developed from ax-
purpose of the test requires a different design. “Must” is only
enic cultures. The word implies know biota (2). The micro-
used in connection with factors that directly relate to the
cosms described here are not gnotobiotic because of the
acceptability of the test (see Section 17). “Should” is used to
bacteria and other microbes are not known. An organism
state that the specified condition is recommended and ought to
growing “without neighbors” is axenic (that is, free of all
be met in most tests. Although a violation of one “should” is
contaminants); growing with one organism is monoxenic (that
rarely a serious matter, violation of several will often render the
is, the rotifers growing with one species of food bacteria);
results questionable. Terms such as “is desirable,” “is often
growing with two organisms is dixenic; growing with many
desirable,” and “might be desirable” are used in connection
organisms (provided the organisms are known) is gnotobiotic.
with less important factors. “May” is used to mean “is (are)
A culture or community with many undefined organisms can be
allowed to,” “can” is used to mean “is (are) able to,” and
termed “xenic.” The aquatic microcosms used in this practice
“might” is used to mean “could possibly.” Thus, the classic
are xenic because the bacterial component is undefined and
distinction between “may” and “can” is preserved, and “might”
contaminating organisms can enter. (Definitions are in accor-
is never used as a synonym for either “may” or “can.”
dance with (1, 2)).
3.2 For definitions of other terms used in this practice, refer
3.3.8 grazer, n—an animal that grazes or feeds on growing
to Guide E729, Terminology E943, and Guide E1023. For an
plants; in these aquatic communities, organisms that feed on
explanation of units and symbols, refer to IEEE/SI 10.
algae.
3.3 Definitions of Terms Specific to This Standard:
3.3.9 herbivore, n—an animal that feeds on plants, synony-
3.3.1 algal biovolume, n—an estimate of the total volume of
4 3
mous with grazer.
algal cells (×10 μ /mL) (see 14.1.10).
3.3.10 medium, n—the chemical solution (for example,
3.3.2 available algae, n—an estimate of the volume of algae
4 3
T82MV) used in the microcosms.
(×10 μ /mL) presumed available to the Daphnia (see 14.1.10).
3.3.2.1 Discussion—The estimate is calculated from the 3.3.11 microcosm, n—a small ecosystem that is regarded as
numerical abundance of each species of algae, its nominal
miniature or epitome of a large world.
volume, and an availability factor based on its size and growth
3.3.12 primary producer, adj, n—an organism capable of
characteristics (see 14.1.10). Small algal cells are presumed
converting inorganic chemicals and energy into organic com-
100 % available and large, filamentous forms are presumed 1
pounds.
to 20 % available. Species that attach to sediment or walls are
3.3.12.1 Discussion—Primary producers are synonymous
presumed to be less available than planktonic forms.
with autotrophs; in these microcosms they are the algae
3.3.3 axenic, adj—a culture of organisms growing without
(including Cyanobacteria).
neighbors, that is, pure culture free from contaminant organ-
3.3.13 secondary producer, adj, n—an organism that re-
isms (see gnotobiotic (1–2) ).
quires organic chemicals for its energy source.
3.3.13.1 Discussion—Secondary producers are synonymous
with heterotrophs; some researchers define grazers as second-
Boldface numbers in parentheses refer to the list of references at the end of this
practice. ary producers, and carnivores as tertiary producers. In these
E1366 − 23
microcosms, all of the organisms with the exception of the 4.4 The means of the variables are compared between the
algae can be considered secondary producers. control(s) and other treatment(s) to assess the effects of the test
material. A one-way analysis of variance of each variable with
3.3.14 semicontinuous culture, adj, n—a culture that is
accompanying a priori t-tests is performed on data from each
partially harvested and that receives fresh nutrients from time
sampling day. All quantitative data are presented in tables of
to time.
means, standard deviations, and statistical differences. Selected
3.3.14.1 Discussion—Most of the stock algal cultures are
data are displayed in graphics showing the control mean
harvested daily to maintain them in active growth, and are thus
bordered by the “Interval of Nonsignificance” (IND), and the
semicontinuous cultures. A true continuous culture would
treatment means. The findings should describe changes that
require continuous harvesting and a nutrient renewal system.
have been shown on primary, secondary, and ecosystem
3.3.15 treatment, n—the (usually) six replicate microcosms
variables, for example, see Annex A1.
that have had the same (if any) chemical addition; the control
is one treatment.
5. Significance and Use
3.3.16 trophic level, adj, n—refers to position in food chain;
5.1 A microcosm test is conducted to obtain information
useful in analyzing energy flow (3).
concerning toxicity or other effects of a test material on the
3.3.16.1 Discussion—The first trophic level encompasses
interactions among three trophic levels (primary, secondary,
the primary producers; second trophic level encompasses
and detrital) and the competitive interactions within each
grazers or herbivores (sometimes referred to as primary con-
trophic level. As with most natural aquatic ecosystems, the
sumers); third trophic level encompasses carnivores (some-
microcosms depend upon algal production (primary produc-
times referred to as secondary consumers); the fourth trophic
tion) to support the grazer trophic level (secondary
level encompasses top carnivores. The detrital or recycling
production), which along with the microbial community are
level is usually considered a trophic level, but not given a
primarily responsible for the nutrient recycling necessary to
numerical term. These microcosms include the first and second
sustain primary production. Microcosm initial condition in-
trophic levels as well as a detrital (recycling) level.
cludes some detritus (chitin and cellulose) and additional
detritus is produced by the system. The microcosms include
3.3.17 unialgal, adj—refers to an algal culture that contains
only one type (strain, species) of algae, although bacteria or ecologically important processes and organisms representative
of ponds and lakes, but are non-site specific. To the extent
other non-algal species might be present.
possible, all solutions are mixtures of distilled water and
reagent grade chemicals (see Section 8) and all organisms are
4. Summary of Practice
available in commercial culture collections.
4.1 Replicate microcosms are synthesized from a chemi-
5.2 The species used are easy to culture in the laboratory
cally defined medium and sediment which are initially sterile.
On Day 0, 10 species of algae are inoculated and allowed to and some are routinely used for single species toxicity tests
(Guide E729; Practice D3978, Guides E1192 and E1193).
grow in competition with each other. On Day 4, grazers and
detritivores are introduced. On Day 7, an appropriate number Presumably acute toxicity test results with some of these
species would be available prior to the decision to undertake
of the microcosms are selected as being most similar and
randomly assigned to treatments and to specific locations on the microcosm test. If available, single species toxicity results
would aid in distinguishing between indirect and direct effects.
the light table. Test material is added to microcosms in the
appropriate treatments. If the test material is a potential source
5.3 These procedures are based mostly on published meth-
of nutrients, for example, nitrogen, phosphate, or organic
ods (4-6), interlaboratory testing (7-10, 11), intermediate stud-
carbon, another treatment should receive another material that
ies (12-23, 24), statistical studies (25-27) and mathematical
would supply equivalent nutrients. A control treatment is
simulation results (28). Newer studies on jet fuels have been
established and sampled simultaneously with the other treat-
reported (29)(See 15.1 for multivariate statistical analyses) and
ments. If a solvent is used, a solvent control is also established.
on the implications of multispecies testing for pesticide regis-
4.2 All measurements (see 11.5) are collected twice a week tration (30). Environmental Protection Agency, (EPA) and
for the first 28 days (21 days after treatment). Thereafter, Food and Drug Administration, (FDA) published similar mi-
measurements are made twice a week for organism crocosm tests (31). The methods described here were used to
enumerations, 3-point oxygen concentrations, in vivo determine the criteria for Acceptable Tests (Section 16).
fluorescence, pH and absorbance until the end of the Additional papers have been published using this method for
experiment, usually Day 63 (56 days after treatment). After measuring chemical stress on organisms (32).
Day 28, dissolved nutrients (nitrate, phosphate, nitrite, and
5.4 Concurrent to measuring the ecological effects, it is
ammonia) are measured once a week until the end of the
advisable to measure the concentration of the parent test
experiment. Carbon uptake, alkalinity and extracted pigments
chemical, and if possible, the transformation products ((33) see
(chlorophylls, phaeopigment) are measured if results are to be
Section 12). The concentrations can be measured on either the
compared with field studies.
same microcosms or on concurrent replicates. Information on
4.3 Organisms are reinoculated (in small numbers) each the chemical concentrations of parent material and transforma-
week to allow reestablishment of populations after temporary tion products would aid in the assessment of chemical
reductions (see 11.6). persistence, exposure, accumulation, and in interpreting, if
E1366 − 23
recovery is associated with chemical degradation or biological would not invalidate a test if controls behave normally (see
adaptation. This protocol deals only with ecological effects, Section 16). Temperature around microcosms should be con-
because the techniques for fate studies are in general usage. tinuously recorded with a device that will continue to function
during a power failure.
5.5 In the microcosm, as in natural ecosystems, a population
6.1.2 Work Surface—The table should be at least 2.6 by 0.85
must be able to obtain its requirements from the products of
m (8 ft 9 in. by 2 ft 9 in.) and have a white or light-colored top
other trophic levels, to maintain a birth rate equal to or greater
or covering.
than its death rate, and to support populations of organisms that
−2
6.1.3 Illumination—80 μE m photosynthetically active
will remove its waste products. As in natural ecosystems,
−1
radiation s (850 to 1000 fc) of warm or cool white light
several organisms might be capable of fulfilling the same
should be provided at the top of the table (see Guide E1733).
function, and shifts in species dominance can occur without
LED lights have been satisfactory for similar microcosms. For
disruption of an ecological process. However, species that are
standard fluorescent tubes, a period of 2 to 3 weeks of use
“ecological equivalents” in one function might not be “equiva-
should be allowed after the installation of new tubes and
lent” in other functions; for example, a filamentous alga and a
ballasts to avoid the initially higher light output. Tubes usually
single cell alga might equally produce O , remove NO , NH ,
2 3 3
are stable for about six months and ballasts for about two years.
and PO , but differ in the type of grazer populations they can
Declining light output might occur in older tubes and ballasts.
sustain, for example, filamentous alga might support amphi-
Light intensity should be measured weekly and recorded. The
pods whereas unicellular algae might support Daphnia.
light meter should be moved over the table top to establish a
5.6 Results of these microcosm tests might be more likely to
light isobar where values are 610 %. The microcosm contain-
be indicative of natural ecosystem responses to chemicals than
ers should be placed within this area in an oval configuration
single species toxicity tests because microcosm tests can
(see Fig. 1). A light cycle of 12 h OFF and 12 h ON should be
indicate the explosive population increases that might occur in
established. Unless the table is enclosed care should be taken
a community when more sensitive competitors or predators are
that other room lights are off during the dark period.
eliminated or the food supply is increased through competitive
6.2 Containers:
interactions. Also, microcosm tests are more likely to display
6.2.1 All containers that might contact stock solutions, test
the effects of chemical transformation or increased exposure to
certain organisms by means of concentration of parent or solutions, or any water into which test organisms will be placed
should not contain substances that can be leached or dissolved
degradation products in their food source or habitat.
by aqueous solutions in amounts that can adversely effect
5.7 A list of potential ecological effects is provided to serve
aquatic organisms. In addition, equipment and facilities that
as a summary (see Annex A1).
contact stock solutions or test solutions should be chosen to
5.8 The microcosm test can also be used to obtain informa-
minimize sorption of test materials from water. Glass, Type
tion on the toxicity or other effects of species or strains, not
316 stainless steel, nylon, and fluorocarbon plastics should be
included in the control inocula (13). Additional modifications
used whenever possible to minimize leaching, dissolution, and
might be required.
sorption, except that stainless steel should not be used for tests
on metals.
5.9 Explicit Limitations of the Aquatic Microcosm Protocol:
5.9.1 The scope of the test is limited in the following 6.2.2 One-gal (3.8-L) Glass Jars—recommended for micro-
respects: cosms; soft glass is satisfactory if new containers are used for
5.9.1.1 No fish or other vertebrates are included, each test. The jars should measure approximately 16.0 cm wide
5.9.1.2 Predation on Daphnia is extremely limited or at the shoulder and be 25 cm tall with a 10.6-cm opening. Jars
absent, should be rinsed with 0.1 N HCl and glass-distilled water
5.9.1.3 The ecosystem becomes nutrient limited, before use.
5.9.1.4 The inocula are not gnotobiotic and aseptic tech-
6.3 Major Equipment Items:
nique is not used (except in maintaining stock cultures of
6.3.1 Autoclave, (large enough to sterilize several micro-
microorganisms). Contaminating microorganisms are likely to
cosm containers, media carboys, glassware, and solutions).
be introduced with the larger organisms and during sampling.
6.3.2 Standard Laboratory Facilities, for preparing
5.9.1.5 Most detrital processing is carried out by the sedi-
solutions, including balances for weighing to tenths and
ment microbial community, but this community is not clearly
hundredths of a gram; volumetric flasks, pipettes, and gradu-
described or measured by this protocol.
ated cylinders.
5.9.2 Extrapolation to natural ecosystems should consider
6.3.3 Compound Microscope, with a 40× water immersion
differences in community structure, limiting factors, and water
objective and an 8× ocular are recommended.
chemistry (see Section 17).
6.3.4 Stereomicroscope, with magnification of 10× to 100×.
6. Apparatus
6.3.5 Fluorometer, (for in vivo fluorescence).
6.3.6 Oxygen Meter, with exchangeable electrodes. (New
6.1 Facilities:
electrodes should be used with each new chemical; control
6.1.1 Temperature Control—An incubator or temperature
electrodes from previous experiments can be reused.)
controlled room is required that provides an environment of
6.3.7 Spectrophotometer.
20 °C to 25 °C with the minimal dimensions of 2.6 by 0.85 by
0.8 m high. Short periods of temperatures outside this range 6.3.8 pH Meter, with sensitivity to at least 0.1 pH units.
E1366 − 23
FIG. 1 Position of Microcosms under Lights (6.2.3 and 12.3.1)
6.3.9 Apparatus for Analysis of Nitrate, Nitrite, Ammonia, laboratory and of the controls. If the organisms are genetically
and Phosphate. engineered, appropriate containment procedures should be
6.3.10 Refrigerator, with freezer for storage of medium used (13, 38). The microcosms can be autoclaved at the
component solutions and samples. conclusion of the test.
6.3.11 Computer, to process the data.
7.4 Cleaning of equipment with a volatile solvent such as
acetone should be performed only in a well-ventilated area in
7. Hazards
which no smoking is allowed and no open flame, such as a pilot
7.1 Material safety data sheets should be reviewed for test
light, is present.
substances and reagents to evaluate the safety hazard. Appro-
7.5 To prepare dilute acid solutions, concentrated acid
priate protective clothing such as laboratory coats, aprons, and
should be added to water, not vice versa. Opening a bottle of
glasses and equipment should be used when conducting this
concentrated acid and mixing it with water should be per-
test.
formed only in a fume hood.
7.1.1 Special precautions, such as covering test chambers
and ventilating the area surrounding the chambers, should be
7.6 Because test solutions are usually good conductors of
taken when conducting tests on volatile materials. Information
electricity, use of ground fault systems and leak detectors
on toxicity to humans (34), recommended handling procedures
should be considered to help avoid electrical shocks.
(35) and chemical and physical properties of the test material
should be studied before a test is begun. Special procedures
8. Microcosm Components
might be necessary with radio-labeled test materials (36) and
8.1 Microcosm Medium—T82MV (Table 1) is recom-
with materials that are, or are suspected of being carcinogenic
mended and was used in the interlaboratory testing experi-
(37).
ments (7-10). Related media used for organism culture main-
7.2 Although disposal of stock solutions, test solutions, and
tenance (see Section 10) are described in Appendix X1.
test organisms poses no special problems in most cases, health
Alternative microcosm medium (T86MVK) with additional
and safety precautions and applicable regulations should be
trace metals is also described (Appendix X1) but has not been
considered before beginning a test. Removal or degradation of
as extensively tested. All of these media are designed to have
test material might be desirable before disposal of stock and
low pH buffer and low metal chelation capacity and some
test solutions.
might be suitable for complete microcosm studies. Media used
7.3 If microorganisms are used as test material, precautions in earlier studies are described in Appendix X1. Related media
might need to be taken to prevent contamination of the are recommended for maintenance of stock cultures (described
E1366 − 23
TABLE 1 Microcosm Medium (T82MV) and Sediment Composition
in Section 10). All of these media can be made by adding
(see 8.1)
various quantities of master solutions to distilled water, such as
Type II or III (Specification D1193).
NOTE 1—Microcosm composition is 3 L of liquid medium and 200.1 g
of sediment (see 8.2 – 8.4 for direction).
8.2 Medium Preparation:
NOTE 2—pH adjusted to 7.0 with 0.1 N HCl.
8.2.1 The medium should be prepared as follows:
Medium T82MV Composition
(1) Read instructions through 8.2.5,
Concentration
Molecular
(2) Prepare master solutions (8.2.2); sterilize if so
Compound
Weight
mM Element mg/L
indicated,
NaNO 85.0 0.5 N 7.0
(3) Prepare final basal medium (8.2.3), autoclave and cool,
MgSO ·7H O 246.5 0.1 Mg 2.43
4 2
(4) Add sterile solutions to final basal medium (8.2.4), and
KH PO 136.0 0.04 P 1.23
2 4
A
(5) Adjust pH (8.2.5). NaOH 40.0 0.032 Na 0.74
CaCl ·2H O 147.0 1.0 Ca 40.0
2 2
8.2.2 Master Solutions—Non-sterile master solutions can be
NaCl 58.5 1.5 Na 34.5
prepared in 1-L bottles and refrigerated prior to use. Sterile
Al (SO ) ·18H O 666.5 0.0048 Al 0.26
2 4 3 2
B
Na SiO ·9H O 284.0 0.80 Na 36.8
master solutions can be stored in serum-capped or screw-top 2 3 2
Si 22.4
containers in the refrigerator. Master solutions are stable and
Trace Metals μM mg/L
can be used for up to a year if prepared and stored satisfacto-
FeSO ·7H O 278.0 1.12 Fe 0.0625
4 2
EDTA 292.0 1.42 EDTA 0.4146
rily. Cloudiness or precipitation indicates the need for replace-
H BO 61.8 0.75 B 0.008
3 3
ment.
ZnSO ·7H O 287.5 0.025 Zn 0.0015
4 2
MnCl ·4H O 197.9 0.25 Mn 0.0135
8.2.2.1 Each of the master solutions (A through K, MV, 10×
2 2
Na MoO ·2H O 242.0 0.025 Mo 0.0024
2 4 2
Silicate and (optional) Keating’s metals) should be prepared
CuSO ·5H O 249.7 0.005 Cu 0.00032
4 2
and stored separately (see Tables 2-4).
Co(NO ) ·6H O 291.0 0.0025 Co 0.00015
3 2 2
C
Murphy’s Vitamins μM mg/L
8.2.2.2 Silicate Solution (10×)—Add 45.95 g Na SiO
2 3
Calcium pantothenate 476.5 1.47 0.70
·9H O to distilled water in a 1-L volumetric flask, filter through
Cyanocobalamin (B ) 1355.4 0.000022 0.00003
a 0.22-μ membrane filter, and store in a sterile nontoxic plastic Thiamin (B ) 337.3 0.18 0.06
Riboflavin (B ) 376.4 0.11 0.04
bottle.
Nicotinamide 122.1 1.06 0.13
8.2.2.3 HCl Solution—Add 100 mL of concentrated HCl
Folic acid 441.4 0.75 0.33
Biotin 244.3 0.12 0.03
with 900 mL of distilled water in a volumetric flask, transfer
Putrescine 161.1 0.19 0.03
the solution to a glass container and autoclave.
Choline 181.7 2.75 0.50
8.2.3 Preparation and Sterilization of Final Basal Medium: Inositol 216.2 5.09 1.10
Pyridoxine monohydrochloride 205.7 2.43 0.50
8.2.3.1 Place 16 L of distilled water in a clean 20-L (5-gal)
Sediment g/microcosm
carboy. Add the solutions listed at the end of this paragraph and
Silica sand 200.0
Chitin 0.5
dilute with distilled water to 18 L. A nontoxic stopper or top
Cellulose powder 0.5
equipped with a serum stopper and a clamped-off dispensing
A
NaOH is added with the KH PO master solution. There are additional minor
2 4
tube is added. Six carboys of medium are needed for a
+
sources of Na (trace metals and Murphy’s vitamins); NaCl and Na SiO -9H 0 are
2 3 2
+ + +
microcosm experiment (if 30 microcosms are initiated).
the major sources of Na . It is important that Na and not K be the major
monovalent cation.
Concentration
B
Master
If diatoms are not used, the Na SiO -9H 0 concentration can be reduced to 0.08
2 3 2
Salt mL/L mL/18 L mM
+
Solution
mM (3.6 mg/L) see Table 2. The NaCl solution will assure that Na is the major
(Final Solution)
monovalent cation in the final medium.
A NaNO 5 90 0.5
C
Murphy’s vitamins (Table 3) were used in the development and testing of the
B MgSO ·7H O 1 18 0.1
4 2
protocol (1-28). More recent work (39-44) has indicated that not all of these organic
D CaCl ·2H O 10 180 1.0
2 2
compounds are needed, at least for algae-Daphnia magna microcosms, if Keat-
E NaCl 15 270 1.5
ing’s Metal Solution of trace metals (Table 4) and 3 vitamins ( B , Biotin, and
H Al (SO ) ·18H O 1 18 0.0048
2 4 3 2
Thiamine) are added. See the footnote B to Table 3 for preparation of the vitamin
I Na SiO ·9H O 5 90 0.080
2 3 2
master mixture.
8.2.3.2 The final basal medium should be dispensed into the
microcosm jars and sterilized with the sediment and allowed to
8.2.4 Addition of sterile solutions to the final basal medium
cool (see 8.4). Alternately, the final basal medium can be
to prepare the medium T86MV and medium T86MVK are as
autoclaved in the carboys (121°C, 60 min), allowed to cool,
follows:
and be dispensed aseptically into sterile microcosm jars. The
Sterile Master Solution mL/L mL/18 L
final basal medium is stable and should not precipitate during
C 0.4 7.2
autoclaving or storage. The final basal medium lacks
K 0.05 0.9
phosphate, trace metals, and vitamins, which are added in the MV 1.0 18.0
Silicate Solution (10×) 5.0 90.0
individual test chambers. The pH is also adjusted in the test
A A
Keating’s Metal Solution 1 18.0
containers.
HCl to pH 7 to pH 7
8.2.3.3 If the medium is being used for the nutrient reservoir
A
Use only for medium T86MVK.
of the algal semicontinuous cultures, the final basal medium
should be autoclaved in the carboy.
E1366 − 23
TABLE 2 Master Solutions
8.4.1 To assemble microcosms, 200 g of silica sand are
Concentration weighed into a beaker, 0.5 g of chitin and 0.5 g of cellulose
Master Molecular
Salt
Solution Weight
g/L M powder are added, then the sediment is placed in the rinsed
A NaNO 85.0 8.5 0.1 microcosm containers. At least 6 extra microcosms with sand,
B MgSO ·7H O 246.5 24.65 0.1
4 2
chitin, and cellulose should be prepared in case of breakage
A
C KH PO 136.0 13.6 0.1
2 4
during autoclaving and to allow culling of outliers (see 11.3).
NaOH 40.0 3.2 0.08
D CaCl ·2H O 147.0 14.7 0.1 Six carboys of unsterilized final basal medium (see 8.2.3) are
2 2
E NaCl 58.5 5.84 0.1
made if 30 microcosms are to be prepared. Five hundred mL of
B
F FeSO ·7H O 278.0 24.9 0.0895
4 2
C media from each carboy are added to each container (for a total
EDTA 292.0 26.1 0.0895
NaOH 40.0 10.7 0.268
of 3 L per container); this ensures that each microcosm
D
G H BO 61.8 1.85 0.03
3 3
receives medium from each carboy to provide uniform initial
ZnSO ·7H O 287.5 0.287 0.001
4 2
conditions.
MnCl ·4H O 197.9 1.98 0.01
2 2
Na MoO ·2H O 242.0 0.242 0.001
2 4 2
CuSO ·5H O 249.7 0.0499 0.0002
A,B
4 2
TABLE 3 Modified Murphy’s Vitamin Solution
Co(NO ) ·6H O 291.0 0.0291 0.0001
3 2 2
Concentration
H Al (SO ) ·18H O 666.5 3.2 0.0048 Molecular
2 4 3 2
Name
Weight
I Na SiO ·9H O 284.0 4.55 0.016
2 3 2 mg/L mM
I (10×) 45.5 0.16
Calcium pantothenate 476.5 700.0 1.47
E
J EDTA 292.0 29.0 0.1
Cyanocobalamin (B ) 1355.4 0.03 0.000022
NaOH 40.0 12.0 0.3
Thiamin (B ) 337.3 60.0 0.18
F 1
K . . . .
Riboflavin (B ) 376.4 40.0 0.11
A
Solution C should be filter-sterilized through 0.22-μ membrane filter or heat- Nicotinamide 122.1 130.0 1.06
sterilized and stored in a flask with a serum stopper in a refrigerator. Folic Acid 441.4 330.0 0.75
B
Solution F is used to prepare Solution K. Biotin 244.3 30.0 0.12
C
Ethylenedinitrolotetraacetic Acid. (Do not use di-sodium or tetra-sodium EDTA; Putrescine 161.1 30.0 0.19
use the ethylenedinitrolotetraacetic acid form.) EDTA is dissolved in 268 mL of 1N Choline 181.7 500.0 2.75
NaOH. The FeSO ·7H O is added and the volume brought to 1 L. The solution is Inositol 216.2 1100.0 5.09
4 2
aerated overnight and stored in a 1-L bottle with ground glass stopper under Pyridoxine (B ) monohydrochloride 205.7 500.0 2.43
refrigeration.
A
Ingredients are added to 1 L of an alkaline solution that can be made by adding
D
Solution G is used to prepare Solution K.
2 pellets (approximately 100 mg each) of NaOH to 1 L of distilled water,
E
Solution J is used to prepare Solution K.
filter-sterilized through 0.22μ -filter and stored in a flask with a serum stopper in a
F
Solution K is made from Solutions F, G, and J where F is 250 mL, G is 500 mL,
refrigerator. This modification omits the calcium acetate, antibiotics, serum, and
J is 60 mL, and distilled H O is 190 mL.
trace metal solution used by Murphy (45); reduces the vitamins to 1/10 concen-
tration in the final medium and substitutes pyridoxine (B ) for the pyridoxal (listed
by Murphy on a typed erratum).
B
Murphy’s vitamins (Table 3) were used in the development and testing of the
protocol (4-28). More recent work (39-44) has indicated that not all of these organic
NOTE 1—The specified amounts of the listed solutions are added to the
compounds are needed, at least for algae-Daphnia magna microcosms, if Keat-
final basal medium after autoclaving and cooling (see 8.2.3). This prevents
ing’s Metal Solution of trace metals (Table 4) and 3 vitamins (B , Biotin, and
precipitation prior to dispensing. The final medium without Keating’s
Thiamine) are added. The vitamin master solution is made by adding 5 mg of Biotin
metals is termed T82MV; with Keating’s metal solution, it is termed and 5 mg of B to distilled water in a 1 liter volumetric flask. In another 1 liter
T86MVK (see Appendix X1 for the relationships among several similar volumetric flask containing approximately 500 ml of distilled water, dissolve 100
mg of Thiamine; add 100 ml of the Biotin and B mixture, and bring the total
media that were used in the development of the test or are used in 12
volume to 1 liter. The final concentrations of this master solution are: Biotin 0.5
organism cultures—see Section 10).
mg/L, Thiamine 100 mg/L, B 0.5 mg/L. Divide the master solution into approxi-
8.2.5 pH Adjustment—A known volume of medium should mately 100 ml aliquots in sterile plastic bags and store in the freezer. Discard the
remaining biotin-B solution. Add 1 ml of the master vitamin solution per liter of
be removed and titrated with HCl to pH 7. Given the volume
final medium; lower concentrations may be adequate.
of the medium remaining, the volume of HCl necessary to
adjust the pH to 7 should be added aseptically, and the final pH
checked. With reduced Na SiO ·9H O concentrations pH ad-
2 3 2
8.4.2 Containers are then covered with foil and autoclaved a
justments are not likely to be needed.
few at a time at 121°C (15-lb steam pressure) for 45 min. When
8.3 Sediment:
the medium is cool, sterile solutions (see 8.2.4) are added, and
8.3.1 The sediment of each microcosm is composed of the pH is adjusted to 7.0 with 0.1N HCl, then foil covers are
silica sand (200 g), ground, crude chitin (0.5), and cellulose
replaced with 150 by 15-mm plastic petri dishes. A laboratory
powder (0.5 g). worksheet, should document the media preparation.
8.3.1.1 Silica Sand—Approximately 4 kg (four 2-lb bags) of
9. Test Material
sand are emptied into a large container, covered with 10 %
concentrated HCl and mixed. After 2 h, the acid is decanted
9.1 General—The test material should be reagent grade or
and the sand rinsed with distilled water until rinse water
better, unless a test on an effluent, a formulation, commercial
reaches pH 7. Sand is then oven-dried, cooled, and weighed.
product, or technical-grade or use-grade material is specifically
8.3.1.2 Chitin—A small amount of crude chitin is rinsed
well in distilled water and air dried. It is then ground for 10 min
Reagent Chemicals, American Chemical Society Specifications, American
in a blender or grinder, then filtered through a 0.4-mm sieve.
Chemical Society, Washington, DC. For Suggestions on the testing of reagents not
Larger pieces are reground. listed by the American Chemical Society, see Annual Standards for Laboratory
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
8.3.1.3 Cellulose Powder—Weighed directly.
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
8.4 Microcosm Assembly: MD.
E1366 − 23
needed. Concentration should be stated as active ingredients approximately 28 days (5). Other water-miscible organic
when possible. Before a test is begun, the following should be solvents such as methanol, ethanol, and acetone might be used
known about the test material: as solvents, but they might stimulate undesirable growth of
9.1.1 Identities and concentrations of major ingredients and microorganisms and acetone is quite volatile. If an organic
major impurities, for example, impurities constituting more solvent is used, it should be reagent grade or better. A
than 1 % of the material, surfactant should not be used in the preparation of a stock
9.1.2 Solubility and stability in the water. solution because it might affect the form and toxicity of the test
9.1.3 An estimate of the lowest concentration of test mate- material in test solutions.
rial that is acutely toxic to some of the microcosm species, for
9.2.4 If a solvent other than distilled water or medium is
example, D. magna and S. capricornutum,
used, (a) at least one solvent control, using solvent from the
9.1.4 Accuracy and precision of the analytical method at
same batch used to make the stock solution, must be included
planned test concentration(s), and
in the test and (b) a medium control must be included in the
9.1.5 Estimate of toxicity to humans and recommended
test. If no solvent other than medium or distilled water is used,
handling procedures (see 7.1).
only the medium control must be included in the test.
9.2.4.1 The concentration of solvent should be the same in
9.2 Stock Solution:
all test solutions that contain test material and in the solvent
9.2.1 In some cases the test material can be added directly to
control.
the microcosm, but usually it is dissolved in a solvent to form
a stock solution that is then added to the microcosm. If a stock
9.2.4.2 If the test contains both a medium control and a
solution is used, the concentration and stability of the test solvent control, the variables measured in the two controls
material in it should be determined before the beginning of the
should be compared (see Section 15, 16.2, 16.3, and Appendix
test. If the test material is subject to photolysis, the stock X2). If statistically significant differences are detected between
solution should be shielded from light.
the two controls, only the solvent control may be used for
assessing the effects of the test material. If no statistically
significant differences are detected, the data from both controls
TABLE 4 Keating’s Metal Solution (Optional, for use in T86MVK
A
should be used for assessing the effects of the test material.
or T85MVK )
Concentration
Molecular
9.3 Nutrient Control—If the test material might serve as a
Name
Weight
mg/L mM
source of nutrient (N, P, or organic carbon), a similar concen-
NaBr 102.89 64.4 0.626
tration of nutrient, possibly as part of a nontoxic chemical,
SrCl ·6H O 266.52 304.00 1.141
2 2
should be one of the treatment groups. Alternatively, the
RbCl 120.92 141.5 1.17
LiCl 42.39 611.0 14.41
nutrient supply may be considered a direct effect of the test
KI 166.00 6.5 0.0392
compound.
SeO 110.96 1.41 0.0127
NH VO 116.94 1.15 0.00984
4 3
A 10. Test Organisms
Add ingredients and bring volume to 1 L with distilled water. Autoclave and store
in a refrigerator in glass container. Modified from (39). This solution includes only
10.1 Algae (added on Day 0 at initial concentration of 10
those trace metals in Keating’s medium that were not already in T82MV.
cells for each algae species) are as follows: (see Fig. 2).
10.1.1 Anabaena cylindrica,
10.1.2 Ankistrodesmus sp.,
9.2.2 Except possibly for tests on hydrolyzable, oxidizable,
10.1.3 Chlamydomonas reinhardi 90,
and reducible materials, the preferred solvent is medium or
distilled water. Sterilization of the stock solution might be 10.1.4 Chlorella vulgaris,
necessary if the test material is subject to microbial transfor-
10.1.5 Lyngbya sp.,
mation. Several techniques have been specifically developed
10.1.6 Nitzschia kutzigiana (Diatom 216),
for preparing aqueous stock solution of slightly soluble mate-
10.1.7 Scenedesmus obliquus,
rials (46). The minimum necessary amount of a strong acid or
10.1.8 Selenastrum capricornutum, (also known as, Raphi-
base may be used in the preparation of an aqueous stock
docelis subcapitata (Korsh.) Nygaard, Komarek et al.; and
solution, but such reagents might affect the pH of test solutions
Pseudokirchneriella subcapitata (Korshikov) Hindak.
appreciably. Use of a more soluble form of the test material,
10.1.9 Stigeoclonium sp., and
such as chloride or sulfate salts of organic amines, sodium or
10.1.10 Ulothrix sp.
potassium salts of phenols and organic acids, and chloride or
nitrate salts of metals, might affect the pH more than use of the
10.2 Animals (added on Day 4 at the initial numbers
necessary minimum amount of a strong acid or base.
indicated in parentheses) are as follows: (see Fig. 3).
9.2.3 If a solvent other than medium or distilled water is
10.2.1 Daphnia magna (16/microcosm),
used, its concentration in test solutions should be kept to a
10.2.2 Hyalella azteca (12/microcosm),
minimum and should be low enough that it does not affect
10.2.3 Cypridopsis or Cyprinotus incongruens or similar
survival or reproduction of any species used in the microcosm.
species (vidua) (6/microcosm),
In spite of its low toxicity to aquatic animals, low volatility,
10.2.4 Hypotrichs [protozoa] (0.1/mL) (optional), and
and high ability to dissolve many organic chemicals, triethyl-
ene glycol must not be used because it has caused low pH after 10.2.5 Philodina acuticornis (rotifer) (0.03/mL).
E1366 − 23
FIG. 2 Algae Used in the Standardized Aquatic Microcosm (10.1)
10.3 Whenever possible, the species just listed should be chemicals spread from other areas, this fact should be noted.
used. These species were selected on the basis of past success- Stock cultures should be protected against exposure to mate-
ful use. The scientific name of the species used should be rials to be tested to prevent adaptation or genetic selection.
verified using an appropriate taxonomic key.
10.5 Algal Culture Maintenance—Algal cultures should be
10.4 Stock cultures should be examined periodically to
maintained on T82-LowSi agar slants under lights and trans-
verify that contamination has not occurred. Stock cultures
ferred at appropriate intervals. Aseptic technique should be
should be maintained in more than one room to minimize the
used with the maintenance of the stock cultures.
risk of a total loss due to such events as a temperature control
10.5.1 T82-LowSi Agar Slants:
malfunction. This can be done with least effort by transferring
NOTE 2—This differs from the microcosm medium T82MV by (1) the
the older culture to an alternate culture room after the new
omission Murphy’s vitamins, (2) the silicate concentration is 0.08 mM
cultures have been inoculated. Stock cultures should not be
instead of 0.8 mM, and (3) the pH adjustment is unnecessary (see Table 5).
maintained solely in rooms where tests are conducted, equip-
ment is cleaned, or toxic materials are handled. Use of volatile 10.5.2 The mixture (complete with solutions C and K) is
chemicals should be avoide
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E1366 − 11 (Reapproved 2016) E1366 − 23
Standard Practice for
Standardized Aquatic Microcosms: Fresh Water
This standard is issued under the fixed designation E1366; 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.
1. Scope
1.1 This practice covers procedures for obtaining data concerning toxicity and other effects of a test material to a multi-trophic
level freshwater community.community, independent of the location of the test.
1.2 These procedures also might be useful for studying the fate of test materials and transformation products, although
modifications and additional analytical procedures might be necessary.
1.3 Modification of these procedures might be justified by special needs or circumstances. Although using appropriate procedures
is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be
comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these
procedures might provide useful information concerning new concepts and procedures for conducting multi-trophic level tests.
1.4 This practice is arranged as follows:
Section
Referenced Documents 2
Terminology 3
Summary of Practice 4
Significance and Use 5
Apparatus 6
Facilities 6.1
Container 6.2
Equipment 6.3
Hazards 7
Microcosm Components 8
Medium 8.1
Medium Preparation 8.2
Sediment 8.3
Microcosm Assembly 8.4
Test Material 9
General 9.1
Stock Solution 9.2
Nutrient Control 9.3
Test Concentration(s) 9.4
Test Organisms 10
Algae 10.1
Animals 10.2
Specificity of Organisms 10.3
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 Feb. 1, 2016June 1, 2023. Published May 2016July 2023. Originally approved in 1990. Last previous edition approved in 20112016 as
E1366 – 11.E1366 – 11(2016). DOI: 10.1520/E1366-11R16.10.1520/E1366-23.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1366 − 23
Sources 10.4
Algal Culture Maintenance 10.5
Animal Culture Maintenance 10.6
Section
Procedure 11
Experimental Design 11.1
Procedure
Inoculation 11.2
Culling 11.3
Addition of Test Material 11.4
Measurements 11.5
Reinoculations 11.6
Analytical Methodology 12
Data Processing 13
Calculations of Variables from Measurements 14
Statistical Analyses 15
Acceptability of Test 16
Interpretation of Results 17
Report 18
Annex Annex A1
Appendices
Relationship of Media Appendix X1
Statistical Guidance Appendix X2
1.5 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.
1.6 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 and healthsafety, health, and environmental practices and determine
the applicability of regulatory limitations prior to use.Specific hazard statements are given in Section 7.
1.7 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:
D1193 Specification for Reagent Water
D3978 Practice for Algal Growth Potential Testing with Pseudokirchneriella subcapitata
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
E1192 Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates,
and Amphibians
E1193 Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests
E1733 Guide for Use of Lighting in Laboratory Testing
IEEE/SI 10 American National Standard for Use of the International System of Units (SI): The Modern Metric System
3. Terminology
3.1 The words “must,” “should,” “may,” “can,” and “might” have very specific meanings in this practice. “Must” is used to
express an absolute requirement, that is, to state that the test ought to be designed to satisfy the specific condition, unless the
purpose of the test requires a different design. “Must” is only used in connection with factors that directly relate to the acceptability
of the test (see Section 17). “Should” is used to state that the specified condition is recommended and ought to be met in most tests.
Although a 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.”
3.2 For definitions of other terms used in this practice, refer to Guide E729, Terminology E943, and Guide E1023. For an
explanation of units and symbols, refer to IEEE/SI 10.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
E1366 − 23
3.3 Definitions of Terms Specific to This Standard:
4 3
3.3.1 algal biovolume, n—an estimate of the total volume of algal cells (×10 μ /mL) (see 14.1.10).
4 3
3.3.2 available algae, n—an estimate of the volume of algae (×10 μ /mL) presumed available to the Daphnia (see 14.1.10).
3.3.2.1 Discussion—
The estimate is calculated from the numerical abundance of each species of algae, its nominal volume, and an availability factor
based on its size and growth characteristics (see 14.1.10). Small algal cells are presumed 100 % available and large, filamentous
forms are presumed 1 to 20 % available. Species that attach to sediment or walls are presumed to be less available than planktonic
forms.
3.3.3 axenic, adj—a culture of organisms growing without neighbors, that is, pure culture free from contaminant organisms (see
gnotobiotic (1–2) ).
3.3.4 community metabolism, n—the oxygen or carbon dioxide balance of the entire community.
3.3.4.1 Discussion—
In this microcosm, community metabolism is estimated by the gain in oxygen during the lighted period (an estimate of net
photosynthesis—P) and the loss of oxygen during the dark period (an estimate of respiration—R). When expressed as a P/R ratio,
a value of >1 indicates that autotrophic processes are dominant; a value of <1 indicates that heterotrophic processes are dominant.
If the difference of P and R are considered (P-R), a positive number indicates autotrophic processes are dominant, and a negative
number indicates heterotrophic processes are dominant. Because P and R often change in the same direction and magnitude, P/R
maybe less sensitive than P or R considered separately.
3.3.5 detritivore, n—an organism that feeds on detritus, dead organic material.
3.3.6 ecosystem, n—a system made up of a community of animals, plants, and bacteria and its interrelated physical and chemical
environment (3).
3.3.7 gnotobiotic, adj—a culture which the exact composition of the organisms is known, down to the presence or absence of
bacteria. Such cultures are developed from axenic cultures. The word implies know biota (2). The microcosms described here are
not gnotobiotic because of the bacteria and other microbes are not known.
3.3.7.1 Discussion—
Such cultures are developed from axenic cultures. The word implies know biota (2). The microcosms described here are not
gnotobiotic because of the bacteria and other microbes are not known. An organism growing “without neighbors” is axenic (that
is, free of all contaminants); growing with one organism is monoxenic (that is, the rotifers growing with one species of food
bacteria); growing with two organisms is dixenic; growing with many organisms (provided the organisms are known) is
gnotobiotic. A culture or community with many undefined organisms can be termed “xenic.” The aquatic microcosms used in this
practice are xenic because the bacterial component is undefined and contaminating organisms can enter. (Definitions are in
accordance with (1, 2)).
3.3.8 grazer, n—an animal that grazes or feeds on growing plants; in these aquatic communities, organisms that feed on algae.
3.3.9 herbivore, n—an animal that feeds on plants, synonymous with grazer.
3.3.10 medium, n—the chemical solution (for example, T82MV) used in the microcosms.
3.3.11 microcosm, n—a small ecosystem that is regarded as miniature or epitome of a large world.
3.3.12 primary producer, adj, n—an organism capable of converting inorganic chemicals and energy into organic compounds.
3.3.12.1 Discussion—
Primary producers are synonymous with autotrophs; in these microcosms they are the algae (including the blue-
greens).Cyanobacteria).
Boldface numbers in parentheses refer to the list of references at the end of this practice.
E1366 − 23
3.3.13 secondary producer, adj, n—an organism that requires organic chemicals for its energy source.
3.3.13.1 Discussion—
Secondary producers are synonymous with heterotrophs; some researchers define grazers as secondary producers, and carnivores
as tertiary producers. In these microcosms, all of the organisms with the exception of the algae can be considered secondary
producers.
3.3.14 semicontinuous culture, adj, n—a culture that is partially harvested and that receives fresh nutrientnutrients from time to
time.
3.3.14.1 Discussion—
Most of the stock algal cultures are harvested daily to maintain them in active growth, and are thus semicontinuous cultures. A
true continuous culture would require continuous harvesting and a nutrient renewal system.
3.3.15 treatment, n—the (usually) six replicate microcosms that have had the same (if any) chemical addition; the control is one
treatment.
3.3.16 trophic level, adj, n—refers to position in food chain; useful in analyzing energy flow (3).
3.3.16.1 Discussion—
The first trophic level encompasses the primary producers; second trophic level encompasses grazers or herbivores (sometimes
referred to as primary consumers); third trophic level encompasses carnivores (sometimes referred to as secondary consumers); the
fourth trophic level encompasses top carnivores. The detrital or recycling level is usually considered a trophic level, but not given
a numerical term. These microcosms include the first and second trophic levels as well as a detrital (recycling) level.
3.3.17 unialgal, adj—refers to an algal culture that contains only one type (strain, species) of algae, although bacteria or other
non-algal species might be present.
4. Summary of Practice
4.1 Replicate microcosms are synthesized from a chemically defined medium and sediment which are initially sterile. On Day 0,
10 species of algae are inoculated and allowed to grow in competition with each other. On Day 4, grazers and detritivores are
introduced. On Day 7, an appropriate number of the microcosms are selected as being most similar and randomly assigned to
treatments and to specific locations on the light table. Test material is added to microcosms in the appropriate treatments. If the
test material is a potential source of nutrients, for example, nitrogen, phosphate, or organic carbon, another treatment should
receive another material that would supply equivalent nutrients. A control treatment is established and sampled simultaneously with
the other treatments. If a solvent is used, a solvent control is also established.
4.2 All measurements (see 11.5) are collected twice a week for the first 28 days (21 days after treatment). Thereafter,
measurements are made twice a week for organism enumerations, 3-point oxygen concentrations, in vivo fluorescence, pH and
absorbance until the end of the experiment, usually Day 63 (56 days after treatment). After Day 28, dissolved nutrients (nitrate,
phosphate, nitrite, and ammonia) are measured once a week until the end of the experiment. Carbon uptake, alkalinity and extracted
pigments (chlorophylls, phaeopigment) are measured if results are to be compared with field studies.
4.3 Organisms are reinoculated (in small numbers) each week to allow reestablishment of populations after temporary reductions
(see 11.6).
4.4 The means of the variables are compared between the control(s) and other treatment(s) to assess the effects of the test material.
A one-way analysis of variance of each variable with accompanying a priori t-tests is performed on data from each sampling day.
All quantitative data are presented in tables of means, standard deviations, and statistical differences. Selected data are displayed
in graphics showing the control mean bordered by the “Interval of Nonsignificance” (IND), and the treatment means. The findings
should describe changes that have been shown on primary, secondary, and ecosystem variables, for example, see Annex A1.
5. Significance and Use
5.1 A microcosm test is conducted to obtain information concerning toxicity or other effects of a test material on the interactions
among three trophic levels (primary, secondary, and detrital) and the competitive interactions within each trophic level. As with
most natural aquatic ecosystems, the microcosms depend upon algal production (primary production) to support the grazer trophic
E1366 − 23
level (secondary production), which along with the microbial community are primarily responsible for the nutrient recycling
necessary to sustain primary production. Microcosm initial condition includes some detritus (chitin and cellulose) and additional
detritus is produced by the system. The microcosms include ecologically important processes and organisms representative of
ponds and lakes, but are non-site specific. To the extent possible, all solutions are mixtures of distilled water and reagent grade
chemicals (see Section 8) and all organisms are available in commercial culture collections.
5.2 The species used are easy to culture in the laboratory and some are routinely used for single species toxicity tests (Guide E729;
Practice D3978, Guides E1192 and E1193). Presumably acute toxicity test results with some of these species would be available
prior to the decision to undertake the microcosm test. If available, single species toxicity results would aid in distinguishing
between indirect and direct effects.
5.3 These procedures are based mostly on previously published methods (4-6), interlaboratory testing (7-10, 11), intermediate
studies (11-12-2223, 24), statistical studies (23-25-2527) and mathematical simulation results (2628). Newer studies on jet fuels
have been reported (2729)(See 15.1 for multivariate statistical analyses) and on the implications of multispecies testing for
pesticide registration (2830). Environmental Protection Agency, (EPA) and Food and Drug Administration, (FDA) published
similar microcosm tests (2931). The methods described here were used to determine the criteria for Acceptable Tests (Section 16).
Additional papers have been published using this method for measuring chemical stress on organisms (32).
5.4 Concurrent to measuring the ecological effects, it is advisable to measure the concentration of the parent test chemical, and
if possible, the transformation products ((3033) see Section 12). The concentrations can be measured on either the same
microcosms or on concurrent replicates. Information on the chemical concentrations of parent material and transformation products
would aid in the assessment of chemical persistence, exposure, accumulation, and in interpreting, if recovery is associated with
chemical degradation or biological adaptation. This protocol deals only with ecological effects, because the techniques for fate
studies are in general usage.
5.5 In the microcosm, as in natural ecosystems, a population must be able to obtain its requirements from the products of other
trophic levels, to maintain a birth rate equal to or greater than its death rate, and to support populations of organisms that will
remove its waste products. As in natural ecosystems, several organisms might be capable of fulfilling the same function, and shifts
in species dominance can occur without disruption of an ecological process. However, species that are “ecological equivalents”
in one function might not be “equivalent” in other functions; for example, a filamentous alga and a single cell alga might equally
produce O , remove NO , NH , and PO , but differ in the type of grazer populations they can sustain, for example, filamentous
2 3 3 4
alga might support amphipods whereas unicellular algae might support Daphnia.
5.6 Results of these microcosm tests might be more likely to be indicative of natural ecosystem responses to chemicals than single
species toxicity tests because microcosm tests can indicate the explosive population increases that might occur in a community
when more sensitive competitors or predators are eliminated or the food supply is increased through competitive interactions. Also,
microcosm tests are more likely to display the effects of chemical transformation or increased exposure to certain organisms by
means of concentration of parent or degradation products in their food source or habitat.
5.7 A list of potential ecological effects is provided to serve as a summary (see Annex A1).
5.8 The microcosm test can also be used to obtain information on the toxicity or other effects of species or strains, not included
in the control inocula (1213). Additional modifications might be required.
5.9 Explicit Limitations of the Aquatic Microcosm Protocol:
5.9.1 The scope of the test is limited in the following respects:
5.9.1.1 No fish or other vertebrates are included,
5.9.1.2 Predation on Daphnia is extremely limited or absent,
5.9.1.3 The ecosystem becomes nutrient limited,
5.9.1.4 The inocula are not gnotobiotic and aseptic technique is not used (except in maintaining stock cultures of microorganisms).
Contaminating microorganisms are likely to be introduced with the larger organisms and during sampling, andsampling.
E1366 − 23
FIG. 1 Position of Microcosms under Lights (6.2.3 and 12.3.1)
5.9.1.5 Most detrital processing is carried out by the sediment microbial community, but this community is not clearly described
or measured by this protocol.
5.9.2 Extrapolation to natural ecosystems should consider differences in community structure, limiting factors, and water
chemistry (see Section 17).
6. Apparatus
6.1 Facilities:
6.1.1 Temperature Control—An incubator or temperature controlled room is required that provides an environment of 20 to
25°C20 °C to 25 °C with the minimal dimensions of 2.6 by 0.85 by 0.8 m high. Short periods of temperatures outside this range
would not invalidate a test if controls behave normally (see Section 16). Temperature around microcosms should be continuously
recorded with a device that will continue to function during a power failure.
6.1.2 Work Surface—The table should be at least 2.6 by 0.85 m (8 ft 9 in. by 2 ft 9 in.) and have a white or light-colored top or
covering.
−2 −1
6.1.3 Illumination—80 μE m photosynthetically active radiation s (850 to 1000 fc) of warm or cool white light should be
provided at the top of the table. A table (see Guide E1733). LED lights have been satisfactory for similar microcosms. For standard
fluorescent tubes, a period of 2 to 3 weeks of use should be allowed after the installation of new tubes and ballasts to avoid the
initially higher light output. Tubes usually are stable for about six months and ballasts for about two years. Declining light output
might occur in older tubes and ballasts. Light intensity should be measured weekly and recorded. The light meter should be moved
over the table top to establish a light isobar where values are 610 %. The microcosm containers should be placed within this area
in an oval configuration (see Fig. 1). A light cycle of 12 h OFF and 12 h ON should be established. Unless the table is enclosed
care should be taken that other room lights are off when lights over the table are off.during the dark period.
6.2 Containers:
E1366 − 23
6.2.1 All containers that might contact stock solutions, test solutions, or any water into which test organisms will be placed should
not contain substances that can be leached or dissolved by aqueous solutions in amounts that can adversely effect aquatic
organisms. In addition, equipment and facilities that contact stock solutions or test solutions should be chosen to minimize sorption
of test materials from water. Glass, Type 316 stainless steel, nylon, and fluorocarbon plastics should be used whenever possible
to minimize leaching, dissolution, and sorption, except that stainless steel should not be used for tests on metals.
6.2.2 One-gal (3.8-L) Glass Jars—recommended for microcosms; soft glass is satisfactory if new containers are used for each test.
The jars should measure approximately 16.0 cm wide at the shoulder and be 25 cm tall with a 10.6-cm opening. Jars should be
rinsed with 10 %0.1 N HCl and glass-distilled water before use.
6.3 Major Equipment Items:
6.3.1 Autoclave, (large enough to sterilize several microcosm containers, media carboys, glassware, and solutions).
6.3.2 Standard Laboratory Facilities, for preparing solutions, including balances for weighing to tenths and hundredths of a gram;
volumetric flasks, pipettes, and graduated cylinders.
6.3.3 Compound Microscope, with a 40× water immersion objective and an 8× ocular are recommended.
6.3.4 Stereomicroscope, with magnification of 10× to 100×.
6.3.5 Fluorometer, (for in vivo fluorescence).
6.3.6 Oxygen Meter, with exchangeable electrodes. (New electrodes should be used with each new chemical; control electrodes
from previous experiments can be reused.)
6.3.7 Spectrophotometer.
6.3.8 pH Meter, with sensitivity to at least 0.1 pH units.
6.3.9 Apparatus for Analysis of Nitrate, Nitrite, Ammonia, and Phosphate.
6.3.10 Refrigerator, with freezer for storage of medium component solutions and samples.
6.3.11 Computer, to process the data.
7. Hazards
7.1 Material safety data sheets should be reviewed for test substances and reagents to evaluate the safety hazard. Appropriate
protective clothing such as laboratory coats, aprons, and glasses and equipment should be used when conducting this test.
7.1.1 Special precautions, such as covering test chambers and ventilating the area surrounding the chambers, should be taken when
conducting tests on volatile materials. Information on toxicity to humans (3134), recommended handling procedures (3235) and
chemical and physical properties of the test material should be studied before a test is begun. Special procedures might be
necessary with radio-labeled test materials (3336) and with materials that are, or are suspected of being carcinogenic (3437).
7.2 Although disposal of stock solutions, test solutions, and test organisms poses no special problems in most cases, health and
safety precautions and applicable regulations should be considered before beginning a test. Removal or degradation of test material
might be desirable before disposal of stock and test solutions.
7.3 If microorganisms are used as test material, precautions might need to be taken to prevent contamination of the laboratory and
of the controls. If the organisms are genetically engineered, appropriate containment procedures should be used (1213, 3538). The
microcosms can be autoclaved at the conclusion of the test.
7.4 Cleaning of equipment with a volatile solvent such as acetone should be performed only in a well-ventilated area in which
no smoking is allowed and no open flame, such as a pilot light, is present.
E1366 − 23
7.5 To prepare dilute acid solutions, concentrated acid should be added to water, not vice versa. Opening a bottle of concentrated
acid and mixing it with water should be performed only in a fume hood.
7.6 Because test solutions are usually good conductors of electricity, use of ground fault systems and leak detectors should be
considered to help avoid electrical shocks.
8. Microcosm Components
8.1 Microcosm Medium—Medium T82MV (Table 1), is recommended on the basis of interlaboratory testing ) is recommended
and was used in the interlaboratory testing experiments (7-10). An alternative Related media used for organism culture
maintenance (see Section 10) are described in Appendix X1. Alternative microcosm medium (T86MVK) with additional trace
metals is also described (Appendix X1),) but has not been as extensively tested. These All of these media are designed to have
low pH buffer and low metal chelation capacity. capacity and some might be suitable for complete microcosm studies. Media used
in earlier studies are described in Appendix X1. Related media are recommended for maintenance of stock cultures (described in
Section 10). All of these media can be made by adding various quantities of master solutions to distilled water, such as Type II
or III (Specification D1193).
8.2 Medium Preparation:
8.2.1 The medium should be prepared as follows:
(1) Read instructions through 8.2.5,
(2) Prepare master solutions (8.2.2); sterilize if so indicated,
(3) Prepare final basal medium (8.2.3), autoclave and cool,
(4) Add sterile solutions to final basal medium (8.2.4), and
(5) Adjust pH (8.2.5).
8.2.2 Master Solutions—Non-sterile master solutions can be prepared in 1-L bottles with ground glass stoppers and refrigerated
prior to use. Sterile master solutions can be stored in serum-capped or screw-top containers in the refrigerator. Master solutions
are stable and can be used for up to a year if prepared and stored satisfactorily. Cloudiness or precipitation indicates the need for
replacement.
8.2.2.1 Each of the master solutions (A through K, MV, 10× Silicate and (optional) Keating’s metals) should be prepared and
stored separately (see Tables 2-4).
8.2.2.2 Silicate Solution (10×)—Add 45.95 g Na SiO
2 3
·9H O to distilled water in a 1-L volumetric flask, filter through a 0.22-μ membrane filter, and store in a sterile nontoxic plastic
bottle.
8.2.2.3 HCl Solution—Add 100 mL of concentrated HCl with 900 mL of distilled water in a volumetric flask, transfer the solution
to a glass container and autoclave.
8.2.3 Preparation and Sterilization of Final Basal Medium:
8.2.3.1 Place 16 L of distilled water in a clean 20-L (5-gal) carboy. Add the solutions listed at the end of this paragraph and dilute
with distilled water to 18 L. A nontoxic stopper or top equipped with a serum stopper and a clamped-off dispensing tube is added.
Six carboys of medium are needed for a microcosm experiment (if 30 microcosms are initiated).
Concentration
Master
Salt mL/L mL/18 L mM
Solution
(Final Solution)
A NaNO 5 90 0.5
B MgSO ·7H O 1 18 0.1
4 2
D CaCl ·2H O 10 180 1.0
2 2
E NaCl 15 270 1.5
H Al (SO ) ·18H O 1 18 0.0048
2 4 3 2
I Na SiO ·9H O 5 90 0.080
2 3 2
8.2.3.2 The final basal medium should be dispensed into the microcosm jars and sterilized with the sediment and allowed to cool
E1366 − 23
(see 8.4). Alternately, the final basal medium can be autoclaved in the carboys (121°C, 60 min), allowed to cool, and be dispensed
aseptically into sterile microcosm jars. The final basal medium is stable and should not precipitate during autoclaving or storage.
The final basal medium lacks phosphate, trace metals, and vitamins, which are added in the individual test chambers. The pH is
also adjusted in the test containers.
8.2.3.3 If the medium is being used for the nutrient reservoir of the algal semicontinuous cultures, the final basal medium should
be autoclaved in the carboy.
TABLE 1 Microcosm Medium (T82MV) and Sediment Composition
(see 8.1)
NOTE 1—Microcosm composition is 3 L of liquid medium and 200.1 g
of sediment (see 8.2 – 8.4 for direction).
NOTE 2—pH adjusted to 7.0 with sterile 10 % 0.1 N HCl.
Medium T82MV Composition
Concentration
Molecular
Compound
Weight
mM Element mg/L
NaNO 85.0 0.5 N 7.0
MgSO ·7H O 246.5 0.1 Mg 2.43
4 2
KH PO 136.0 0.04 P 1.23
2 4
A
NaOH 40.0 0.032 Na 0.74
CaCl ·2H O 147.0 1.0 Ca 40.0
2 2
NaCl 58.5 1.5 Na 34.5
Al (SO ) ·18H O 666.5 0.0048 Al 0.26
2 4 3 2
B
Na SiO ·9H O 284.0 0.80 Na 36.8
2 3 2
Si 22.4
Trace Metals μM mg/L
FeSO ·7H O 278.0 1.12 Fe 0.0625
4 2
EDTA 292.0 1.42 EDTA 0.4146
H BO 61.8 0.75 B 0.008
3 3
ZnSO ·7H O 287.5 0.025 Zn 0.0015
4 2
MnCl ·4H O 197.9 0.25 Mn 0.0135
2 2
Na MoO ·2H O 242.0 0.025 Mo 0.0024
2 4 2
CuSO ·5H O 249.7 0.005 Cu 0.00032
4 2
Co(NO ) ·6H O 291.0 0.0025 Co 0.00015
3 2 2
C
Murphy’s Vitamins μM mg/L
Calcium pantothenate 476.5 1.47 0.70
Cyanocobalamin (B ) 1355.4 0.000022 0.00003
Thiamin (B ) 337.3 0.18 0.06
Riboflavin (B ) 376.4 0.11 0.04
Nicotinamide 122.1 1.06 0.13
Folic acid 441.4 0.75 0.33
Biotin 244.3 0.12 0.03
Putrescine 161.1 0.19 0.03
Choline 181.7 2.75 0.50
Inositol 216.2 5.09 1.10
Pyridoxine monohydrochloride 205.7 2.43 0.50
Sediment g/microcosm
Silica sand 200.0
Chitin 0.5
Cellulose powder 0.5
A
NaOH is added with the KH PO master solution. There are additional minor
2 4
+
sources of Na (trace metals and Murphy’s vitamins); NaCl and Na SiO -9H 0 are
2 3 2
+ + +
the major sources of Na . It is important that Na and not K be the major
monovalent cation.
B
If diatoms are not used, the Na SiO -9H 0 concentration can be reduced to 0.08
2 3 2
+
mM (3.6 mg/L) see Table 2. The NaCl solution will assure that Na is the major
monovalent cation in the final medium.
C
Murphy’s vitamins (Table 3) were used in the development and testing of the
protocol (1-2628). More recent work (36-39-4144) has indicated that not all of
these organic compounds are needed, at least for algae-Daphniaalgae-Daphnia
magna microcosms, if Keating’s Metal Solution of trace metals (Table 4) and 3
vitamins ( B , Biotin, and Thiamine) are added. See the footnote B to Table 3 for
preparation of the vitamin master mixture.
8.2.4 Addition of sterile solutions to the final basal medium to prepare the medium T86MV and medium T86MVK are as follows:
E1366 − 23
Sterile Master Solution mL/L mL/18 L
C 0.4 7.2
K 0.05 0.9
MV 1.0 18.0
Silicate Solution (10×) 5.0 90.0
A A
Keating’s Metal Solution 1 18.0
HCl to pH 7 to pH 7
A
Use only for medium T86MVK.
TABLE 2 Master Solutions
Concentration
Master Molecular
Salt
Solution Weight
g/L M
A NaNO 85.0 8.5 0.1
B MgSO ·7H O 246.5 24.65 0.1
4 2
A
C KH PO 136.0 13.6 0.1
2 4
NaOH 40.0 3.2 0.08
D CaCl ·2H O 147.0 14.7 0.1
2 2
E NaCl 58.5 5.84 0.1
B
F FeSO ·7H O 278.0 24.9 0.0895
4 2
C
EDTA 292.0 26.1 0.0895
NaOH 40.0 10.7 0.268
D
G H BO 61.8 1.85 0.03
3 3
ZnSO ·7H O 287.5 0.287 0.001
4 2
MnCl ·4H O 197.9 1.98 0.01
2 2
Na MoO ·2H O 242.0 0.242 0.001
2 4 2
CuSO ·5H O 249.7 0.0499 0.0002
4 2
Co(NO ) ·6H O 291.0 0.0291 0.0001
3 2 2
H Al (SO ) ·18H O 666.5 3.2 0.0048
2 4 3 2
I Na SiO ·9H O 284.0 4.55 0.016
2 3 2
I (10×) 45.5 0.16
E
J EDTA 292.0 29.0 0.1
NaOH 40.0 12.0 0.3
F
K . . . .
A
Solution C should be filter-sterilized through 0.22-μ membrane filter or heat-
sterilized and stored in a flask with a serum stopper in a refrigerator.
B
Solution F is used to prepare Solution K.
C
Ethylenedinitrolotetraacetic Acid. (Do not use di-sodium or tetra-sodium EDTA;
use the ethylenedinitrolotetraacetic acid form.) EDTA is dissolved in 268 mL of 1N
NaOH. The FeSO ·7H O is added and the volume brought to 1 L. The solution is
4 2
aerated overnight and stored in a 1-L bottle with ground glass stopper under
refrigeration.
D
Solution G is used to prepare Solution K.
E
Solution J is used to prepare Solution K.
F
Solution K is made from Solutions F, G, and J where F is 250 mL, G is 500 mL,
J is 60 mL, and distilled H O is 190 mL.
E1366 − 23
NOTE 1—The specified amounts of the listed solutions are added to the final basal medium after autoclaving and cooling (see 8.2.3). This prevents
precipitation prior to dispensing. The final medium without Keating’s metals is termed T82MV; with Keating’s metal solution, it is termed T86MVK (see
Appendix X1 for the relationships among several similar media that were used in the development of the test or are used in organism cultures—see Section
10).
8.2.5 pH Adjustment—A known volume of medium should be removed and titrated with HCl to pH 7. Given the volume of the
medium remaining, the volume of HCl necessary to adjust the pH to 7 should be added aseptically, and the final pH checked. With
reduced Na SiO ·9H O concentrations pH adjustments are not likely to be needed.
2 3 2
8.3 Sediment:
8.3.1 The sediment of each microcosm is composed of the silica sand (200 g), ground, crude chitin (0.5), and cellulose powder
(0.5 g).
8.3.1.1 Silica Sand—Approximately 4 kg (four 2-lb bags) of sand are emptied into a large container, covered with 10 %
concentrated HCl and mixed. After 2 h, the acid is decanted and the sand rinsed with distilled water until rinse water reaches pH
7. Sand is then oven-dried, cooled, and weighed.
8.3.1.2 Chitin—A small amount of crude chitin is rinsed well in distilled water and air dried. It is then ground for 10 min in a
blender or grinder, then filtered through a 0.4-mm sieve. Larger pieces are reground.
8.3.1.3 Cellulose Powder—Weighed directly.
8.4 Microcosm Assembly:
8.4.1 To assemble microcosms, 200 g of silica sand are weighed into a beaker, 0.5 g of chitin and 0.5 g of cellulose powder are
added, then the sediment is placed in the rinsed microcosm containers. At least 6 extra microcosms with sand, chitin, and cellulose
should be prepared in case of breakage during autoclaving and to allow culling of outliers (see 11.3). Six carboys of unsterilized
final basal medium (see 8.2.3) are made if 30 microcosms are to be prepared. Five hundred mL of media from each carboy are
added to each container (for a total of 3 L per container); this ensures that each microcosm receives medium from each carboy
to provide uniform initial conditions.
8.4.2 Containers are then covered with foil and autoclaved a few at a time at 121°C (15-lb steam pressure) for 45 min. When the
medium is cool, sterile solutions (see 8.2.4) are added, and pH is adjusted to 7.0 with 10 %0.1N HCl, then foil covers are replaced
with 150 by 15-mm plastic petri dishes. A laboratory worksheet, should document the media preparation.
9. Test Material
9.1 General—The test material should be reagent grade or better, unless a test on an effluent, a formulation, commercial product,
or technical-grade or use-grade material is specifically needed. Concentration should be stated as active ingredients when possible.
Before a test is begun, the following should be known about the test material:
9.1.1 Identities and concentrations of major ingredients and major impurities, for example, impurities constituting more than 1 %
of the material,
9.1.2 Solubility and stability in the water.
9.1.3 An estimate of the lowest concentration of test material that is acutely toxic to some of the microcosm species, for example,
D. magna and S. capricornutum,
9.1.4 Accuracy and precision of the analytical method at planned test concentration(s), and
9.1.5 Estimate of toxicity to humans and recommended handling procedures (see 7.1).
Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For Suggestions on the testing of reagents not listed by
the American Chemical Society, see Annual Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National
Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
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A,B
TABLE 3 Modified Murphy’s Vitamin Solution
Concentration
Molecular
Name
Weight
mg/L mM
Calcium pantothenate 476.5 700.0 1.47
Cyanocobalamin (B ) 1355.4 0.03 0.000022
Thiamin (B ) 337.3 60.0 0.18
Riboflavin (B ) 376.4 40.0 0.11
Nicotinamide 122.1 130.0 1.06
Folic Acid 441.4 330.0 0.75
Biotin 244.3 30.0 0.12
Putrescine 161.1 30.0 0.19
Choline 181.7 500.0 2.75
Inositol 216.2 1100.0 5.09
Pyridoxine (B ) monohydrochloride 205.7 500.0 2.43
A
Ingredients are added to 1 L of an alkaline solution that can be made by adding
2 pellets (approximately 100 mg each) of NaOH to 1 L of distilled water,
filter-sterilized through 0.22μ -filter and stored in a flask with a serum stopper in a
refrigerator. This modification omits the calcium acetate, antibiotics, serum, and
trace metal solution used by Murphy (4245); reduces the vitamins to 1/10
concentration in the final medium and substitutes pyridoxine (B ) for the pyridoxal
(listed by Murphy on a typed erratum).
B
Murphy’s vitamins (Table 3) were used in the development and testing of the
protocol (4-2628). More recent work (36-39-4144) has indicated that not all of
these organic compounds are needed, at least for algae-Daphnia magna
microcosms, if Keating’s Metal Solution of trace metals (Table 4) and 3 vitamins
(B , Biotin, and Thiamine) are added. The vitamin master solution is made by
adding 5 mg of Biotin and 5 mg of B to distilled water in a 1 liter volumetric flask.
In another 1 liter volumetric flask containing approximately 500 ml of distilled water,
dissolve 100 mg of Thiamine; add 100 ml of the Biotin and B mixture, and bring
the total volume to 1 liter. The final concentrations of this master solution are: Biotin
0.5 mg/L, Thiamine 100 mg/L, B 0.5 mg/L. Divide the master solution into
approximately 100 ml aliquots in sterile plastic bags and store in the freezer.
Discard the remaining biotin-B solution. Add 1 ml of the master vitamin solution
per liter of final medium; lower concentrations may be adequate.
9.2 Stock Solution:
9.2.1 In some cases the test material can be added directly to the microcosm, but usually it is dissolved in a solvent to form a stock
solution that is then added to the microcosm. If a stock solution is used, the concentration and stability of the test material in it
should be determined before the beginning of the test. If the test material is subject to photolysis, the stock solution should be
shielded from light.
TABLE 4 Keating’s Metal Solution (Optional, for use in T86MVK
A
or T85MVK )
Concentration
Molecular
Name
Weight
mg/L mM
NaBr 102.89 64.4 0.626
SrCl ·6H O 266.52 304.00 1.141
2 2
RbCl 120.92 141.5 1.17
LiCl 42.39 611.0 14.41
KI 166.00 6.5 0.0392
SeO 110.96 1.41 0.0127
NH VO 116.94 1.15 0.00984
4 3
A
Add ingredients and bring volume to 1 L with distilled water. Autoclave and store
in a refrigerator in glass container. Modified from (3639). This solution includes
only those trace metals in Keating’s medium that were not already in T82MV.
9.2.2 Except possibly for tests on hydrolyzable, oxidizable, and reducible materials, the preferred solvent is medium or distilled
water. Sterilization of the stock solution might be necessary if the test material is subject to microbial transformation. Several
techniques have been specifically developed for preparing aqueous stock solution of slightly soluble materials (4346). The
minimum necessary amount of a strong acid or base may be used in the preparation of an aqueous stock solution, but such reagents
might affect the pH of test solutions appreciably. Use of a more soluble form of the test material, such as chloride or sulfate salts
of organic amines, sodium or potassium salts of phenols and organic acids, and chloride or nitrate salts of metals, might affect the
pH more than use of the necessary minimum amount of a strong acid or base.
E1366 − 23
9.2.3 If a solvent other than medium or distilled water is used, its concentration in test solutions should be kept to a minimum and
should be low enough that it does not affect survival or reproduction of any species used in the microcosm. In spite of its low
toxicity to aquatic animals, low volatility, and high ability to dissolve many organic chemicals, triethylene glycol must not be used
because it has caused low pH after approximately 28 days (5). Other water-miscible organic solvents such as methanol, ethanol,
and acetone might be used as solvents, but they might stimulate undesirable growth of microorganisms and acetone is quite
volatile. If an organic solvent is used, it should be reagent grade or better. A surfactant should not be used in the preparation of
a stock solution because it might affect the form and toxicity of the test material in test solutions.
9.2.4 If a solvent other than distilled water or medium is used, (a) at least one solvent control, using solvent from the same batch
used to make the stock solution, must be included in the test and (b) a medium control must be included in the test. If no solvent
other than medium or distilled water is used, only the medium control must be included in the test.
9.2.4.1 The concentration of solvent should be the same in all test solutions that contain test material and in the solvent control.
9.2.4.2 If the test contains both a medium control and a solvent control, the variables measured in the two controls should be
compared (see Section 15, 16.2, 16.3, and Appendix X2). If statistically significant differences are detected between the two
controls, only the solvent control may be used for assessing the effects of the test material. If no statistically significant differences
are detected, the data from both controls should be used for assessing the effects of the test material.
9.3 Nutrient Control—If the test material might serve as a source of nutrient (N, P, or organic carbon), a similar concentration of
nutrient, possibly as part of a nontoxic chemical, should be one of the treatment groups. Alternatively, the nutrient supply may be
considered a direct effect of the test compound.
10. Test Organisms
10.1 Algae (added on Day 0 at initial concentration of 10 cells for each algae species) are as follows: (see Fig. 2).
10.1.1 Anabaena cylindrica,
10.1.2 Ankistrodesmus sp.,
10.1.3 Chlamydomonas reinhardi 90,
10.1.4 Chlorella vulgaris,
10.1.5 Lyngbya sp.,
10.1.6 Nitzschia kutzigiana (Diatom 216),
10.1.7 Scenedesmus obliquus,
10.1.8 Selenastrum capricornutum, (also known as, Raphidocelis subcapitata (Korsh.) Nygaard, Komarek et al.; and Pseudokirch-
neriella subcapitata (Korshikov) Hindak.
10.1.9 Stigeoclonium sp., and
10.1.10 Ulothrix sp.
10.2 Animals (added on Day 4 at the initial numbers indicated in parentheses) are as follows: (see Fig. 3).
10.2.1 Daphnia magna (16/microcosm),
10.2.2 Hyalella azteca (12/microcosm),
10.2.3 Cypridopsis or Cyprinotus incongruens or similar species (vidua) (6/microcosm),
10.2.4 Hypotrichs [protozoa] (0.1/mL) (optional), and
E1366 − 23
FIG. 2 Algae Used in the Standardized Aquatic Microcosm (10.1)
10.2.5 Philodina acuticornis (rotifer) (0.03/mL).
10.3 Whenever possible, the species just listed should be used. These species were selected on the basis of past successful use.
The scientific name of the species used should be verified using an appropriate taxonomic key.
10.4 Stock cultures should be examined periodically to verify that contamination has not occurred. Stock cultures should be
maintained in more than one room to minimize the risk of a total loss due to such events as a temperature control malfuncti
...