United States Prevention, Pesticides EPA712-C-96-164
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.5400
Algal Toxicity, Tiers I and
'Public Draft"
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.5400 Algal toxicity, Tiers I and II.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1050 Algal Acute Toxicity
Test; OPP 123-2 Growth and Reproduction of Aquatic Plants (Tier 2)
(Pesticide Assessment Guidelines, Subdivision J—Hazard Evaluation;
Nontarget Plants) EPA report 540/09-82-020, 1982; and OECD 201, Algal
Growth Inhibition Test.
(b) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures (chemicals) sub-
ject to environmental effects test regulations, and was written specifically
for Selenastrum capricornutum and Skeletonema costatum (see paragraph
(d)(2)(iii) of this guideline). Use of Anabaena flos-aquae or Navicula
pelliculosa may require some specific modifications in test procedures.
This guideline prescribes test procedures and conditions using freshwater
and marine algae to develop data on the phytotoxicity of chemicals. EPA
will use data from these tests in assessing the hazard of a chemical to
the environment.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this guideline:
Algicidal means having the property of killing algae.
Algistatic means having the property of inhibiting algal growth.
ECX means the experimentally derived chemical concentration that
is calculated to effect X percent of the test criterion.
Growth means an increase in an algal population based on the number
and/or weight of algal cells per volume of nutrient medium or test solution
in a specified period of time.
Static system means for this test a system in which old nutrient me-
dium (test solution) is not renewed or replaced during the period of the
test.
(d) Test procedures—(1) Summary of the test, (i) In preparation
for the test, fill test containers with appropriate volumes of nutrient me-
dium and/or test solution. Start the test by introducing algae into the test
and control containers in the growth chambers. Environmental conditions
within the growth chambers are established at predetermined limits.
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(ii) At the end of 96 h, and at the end of 24, 48, and 72 h if possible,
enumerate the algal cells in all containers to determine inhibition or stimu-
lation of growth and the pattern of growth in test containers compared
to controls. Use data to define the concentration-response curve, and cal-
culate the EC50 value at these times.
(2) Range-finding test, (i) A range-finding test should be conducted
to determine if definitive testing is necessary, and if so, test chemical con-
centrations for the definitive test. Water solubility of the test chemical (as
well as other physical chemical characteristics, e.g. volatility) should be
determined before definitive testing. A validated analytical method should
also be developed prior to any toxicity testing.
(ii) Algae are exposed to a widely spaced (e.g. log interval) chemical
concentration series. The lowest value in the series, exclusive of controls,
should be at the chemical's detection limit. The upper value, for water
soluble compounds, should be the saturation concentration. A minimum
of three replicates is required: Nominal concentrations of the chemical are
acceptable if it is determined that definitive testing is not required.
(iii) The test is performed once for each of the recommended algal
species or selected alternates. Test chambers should contain equal volumes
of test solution and approximately 1 x 104 S. capricornutum (misidentified
previously as as Monoraphidium capricornutum, correctly also known as
Raphidocelis subcapitata Korsikov), TV. pelliculosa, or A. flos-aquae cells
per milliliter or 7.7xl04 S. costatum cells per milliliter of test solution.
The algae should be exposed to each concentration of test chemical for
up to 96 h.
(iv) Definitive testing may not be necessary if the highest chemical
concentration tested (water saturation concentration or 1,000 mg/L) results
in less than a 50 percent reduction in growth. However, if the lowest con-
centration tested (analytical detection limit) results in greater than a
50 percent reduction in growth definitive testing is necessary. If testing
a pesticide under FIFRA at the maximal labeled dosage, a minimum of
three replicates for each test chemical concentration is required.
(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine the concentration response curves, the EC50's for algal growth for
each species tested, with a minimum amount of testing beyond the range-
finding test.
(ii) Algae should be exposed to five or more concentrations of the
test chemical in a geometric series in which the ratio is between 1.5 and
2.0 (e.g. 2, 4, 8, 16, 32, and 64 mg/L). Often it is possible to choose
test chemical concentrations based on the anticipated slope of the con-
centration-response curve, and these concentrations should bracket the ex-
pected test end-points. Algae are to be placed in a minimum of three rep-
licate test containers for each concentration of test chemical and control.
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With the exception of the use of four replicates for TV. pelliculosa, more
than three replicates may be required to provide sufficient data. Each test
chamber should contain equal volumes of test solution and approximately
1 x 104 Selenastrum, Navicula, or Anabaena cells per milliliter or 7.7x 104
Skeletonema cells per milliliter of test solution. The chemical concentra-
tions should result in greater than 90 percent of algal growth being inhib-
ited or stimulated at the highest concentrations of test substance compared
to controls or that the test concentrations should bracket the expected EC50
value.
(iii) Every test is to include a control (negative control) consisting
of the same nutrient medium, conditions, procedures, and algae from the
same culture, except that none of the test substance is added. If a carrier
is present in any of the test chambers, a separate carrier control is required.
(iv) Positive controls using zinc chloride as a reference chemical
should also be run periodically. The purpose of a positive control with
a reference chemical is to determine that the test algae are responding
to a known chemical in the expected manner. If the algae are responding
to subsequent reference chemical tests consistently, it is assumed that the
algae will respond to other chemicals consistently. Changes in algal re-
sponse caused by such factors as poor nutrition, genetic drift, and contami-
nants may not be detected by negative controls, yet may still influence
test results. At least three concentrations of the reference chemical are run
at or near the expected median effect level.
(v) The test begins when algae (inocula) from 3- to 7-day-old stock
cultures are placed in the test chambers containing test solutions having
the appropriate concentrations of the test substance. The mean cell volume
of inocula should be approximately 35-45 (ig/m3 at the onset of testing.
Algal growth in controls should reach the logarithmic growth phase by
96 h (at which time the number of algal cells should be approximately
1.5xl06/mL for Skeletonema or 3.5xl06/mL for Selenastrum. If loga-
rithmic growth cannot be demonstrated, the test is to be repeated. At the
end of 96 h, and, if possible, at the end of 24, 48, and 72 h, the algal
growth response (number or weight of algal cells per milliliter) in all test
containers and controls is to be determined by an indirect
(spectrophotometry, electronic cell counters, dry weight, etc.) or a direct
(actual microscopic cell count of at least 400 cells per flask) method. Indi-
rect methods are to be calibrated by a direct microscopic count or data
should be presented that relate electronic counts with microscopic counts.
The percentage inhibition or stimulation of growth for each concentration,
EC50, and the concentration-response curves are determined from these
counts.
(vi) A particle counter or microscopic counting cannot be used for
Anabaena unless the filaments are broken up and dispersed using a sy-
ringe, ultrasonic bath, or blender. Limited use of sonification is allowed
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for Anabaena. The procedure used to break up the filaments should result
in consistent filament lengths across treatments and replicates.
Sonification, ultrasonic bath, blender, syringe, or any other methods of
cell separation, other than manual or rotary shaking are not allowed for
Selenastrum, Skeletonema, or Navicula.
(vii) At the end of the definitive test, the following additional analyses
of algal growth response are to be performed:
(A) Determine whether the altered growth response between controls
and test algae (in highest test chemical concentrations) was due to a change
in relative cell numbers, cell sizes, or both. Also note any unusual cell
shapes, color differences, differences in chloroplast morphology,
flocculations, adherence of algae to test containers, or aggregation of algal
cells. These observations are qualitative and descriptive, and are not used
in end-point calculations. They can be useful in determining additional
effects of tested chemicals.
(B) In test concentrations where growth is maximally inhibited,
algistatic effects may be differentiated from algicidal effects by the follow-
ing two methods.
(7) Add 0.5 mL of a 0.1 percent solution (weight/volume) of Evans
blue stain to a 1-mL aliquot of algal suspension from a control container
and to a 1-mL aliquot of algae from the test container having the lowest
concentration of test chemical which completely inhibited algal growth (if
algal growth was not completely inhibited, select an aliquot of algae for
staining from the test container having the highest concentration of test
chemical which inhibited algal growth). Wait 10 to 30 min, examine mi-
croscopically, and determine the percent of the cells which stain blue (indi-
cating cell mortality). A staining control is to be performed concurrently
using heat-killed or formaldehyde-preserved algal cells; 100 percent of
these cells should stain blue.This method will work for Skeletonema and
possibly Navicula, but probably will not work with Selenastrum or
Anabaena.
(2) Remove 0.5 mL aliquots of test solution containing growth-inhib-
ited algae from each replicate test container having the concentration of
test substance evaluated in paragraph (d)(3)(vii)(B)(7) of this guideline.
Combine these aliquots into a new test container and add a sufficient vol-
ume of fresh nutrient medium to dilute the test chemical to a concentration
which does not affect growth. Incubate this subculture under the environ-
mental conditions used in the definitive test for a period of up to 9 days,
and observe periodically (e.g. every other day) for algal growth (direct
or indirect methods) to determine if the algistatic effect noted after the
96-h test is reversible. This subculture test may be discontinued as soon
as growth occurs.
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(4) Analytical measurements—(i) Chemical. (A) Water of sufficient
quality (e.g. ASTM Type I water) is to be used in the preparation of the
nutrient medium. The pH of the test solution and controls is to be meas-
ured at the beginning and at the end of the definitive test. The concentra-
tion of test chemical in the test containers is to be determined at the begin-
ning and end of the definitive test by standard analytical methods which
have been validated prior to the test. An analytical method is unacceptable
if likely degradation products of the chemical, such as hydrolysis and oxi-
dation products, give positive or negative interference to the method. To
be acceptable, the analytical method must be corrected for these inter-
ferences.
(B) At the end of the test and after aliquots have been removed for
algal growth-response determinations, microscopic examination, mortal
staining, or subculturing, the replicate test containers for each chemical
concentration may be pooled into one sample. An aliquot of the pooled
sample may then be taken and the concentration of test chemical deter-
mined after all algal cells have been removed. In addition, the concentra-
tion of test chemical associated with the algae alone may be determined.
Separate and concentrate the algal cells from the test solution by
centrifuging or filtering the remaining pooled sample and measure the test
substance concentration in the algal-cell concentrate.
(ii) Numerical. Algal growth response (as percent of inhibition or
stimulation in the test solutions compared to the controls) is calculated
at the end of the test. Mean and standard deviation should be calculated
and plotted for each treatment and control. Appropriate statistical analyses
(see paragraphs (g)(l) and (g)(2) of this guideline) should provide a good-
ness-of-fit determination for the concentration response curves. The con-
centration response curves are plotted using the mean measured test solu-
tion concentrations obtained in the test chambers at the end of the test.
Results from the recovery phase are used to determine the algistatic con-
centration (refer to paragraph (g)(3) of this guideline). Various statistical
procedures for modeling continuous toxicity data are available and can
be used (see paragraph (g)(4) of this guideline).
(e) Test conditions—(1) Test species. Species of algae recommended
as test organisms for this test are the freshwater green alga, S.
capricornutum (or R. subcapitata), the marine diatom, S. costatum, the
freshwater diatom, TV. pelliculosa, and the blue-green alga or
cyanobacterium, A. flos-aquae. Algae to be used in acute toxicity tests
may be initially obtained from commercial sources and subsequently cul-
tured using sterile technique. Toxicity testing should not be performed
until algal cultures are shown to be actively growing (i.e. capable of loga-
rithmic growth within the test period) in at least two subcultures lasting
7 days each prior to the start of the definitive test. All algae used for
a particular test should be from the same source and the same stock cul-
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ture. Also, the clone of all species should be specified. Test algae should
not have been used in a previous test, either in a treatment or a control.
(2) Facilities—(i) General. (A) Facilities needed to perform this test
include: A growth chamber or a controlled environment room that can
hold the test containers and will maintain the air temperature, lighting in-
tensity, and photoperiod specified in this test guideline, apparatus for cul-
turing and enumerating algae, a source of water of acceptable quality, and
apparatus for carrying out analyses of the test chemical.
(B) Disposal facilities should be adequate to accommodate spent
glassware, algae, and test solutions at the end of the test, and any bench
covering, lab clothing, or other contaminated materials.
(ii) Test containers. Erlenmeyer flasks should be used for test con-
tainers. The flasks may be of any volume between 125 and 500 mL as
long as the same size is used throughout a test and the test solution volume
does not exceed 50 percent of the flask volume.
(iii) Cleaning and sterilization. New test containers may contain
substances which inhibit growth of algae. They are therefore to be cleaned
thoroughly and used several times to culture algae before being used in
toxicity testing. All glassware used in algal culturing or testing is to be
cleaned and sterilized prior to use according to standard good laboratory
practices. These practices include rinsing in a solvent followed by rinsing
with acid.
(iv) Conditioning. Test containers should be conditioned, if needed,
by a rinse with the appropriate test solutions prior to the start of the test.
Decant and add fresh test solutions after an appropriate conditioning period
for the test chemical.
(v) Nutrient medium. (A) Formulation and sterilization of nutrient
medium used for algal culture and preparation of test solutions should con-
form to those currently recommended by the EPA for freshwater and ma-
rine algal bioassays under paragraph (g)(l) of this guideline. Chelating
agents are included in the nutrient medium for optimum cell growth. No
chelating agents are to be included in the nutrient medium used for test
solution preparation if it is suspected that the chelater will interact with
the test chemical. For some chemicals, the stock solution must be prepared
with dilution water (e.g. ASTM Type I water). Volumetric addition should
be limited to prevent dilution of the nutrients. Nutrient medium should
be freshly prepared for algal testing and may be dispensed in appropriate
volumes in test containers and sterilized by autoclaving or filtering by
using a clean water source. The pH of the nutrient medium is to be
7.5 (±0.1) for Selenastrum, 8.1 (±0.1) for Skeletonema, 7.5 (±0.1) for
Navicula, and 7.5 (±0.1) for Anabaena at the start of the test and may
be adjusted prior to test chemical addition with 0.1N or IN NaOH or HC1.
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(B) Dilution water used for preparation of nutrient medium and test
solutions should be of sufficient quality (e.g. ASTM Type I water). Salt-
water for marine algal nutrient medium and test solutions should be pre-
pared by adding a commercial synthetic sea salt formulation or a modified
synthetic seawater formulation to distilled/deionized water to a concentra-
tion of 30 ppt (24 to 35 g/kg).
(vi) Carriers. Nutrient medium is to be used in making stock solu-
tions of the test chemical. If a carrier (or solvent) other than nutrient me-
dium is absolutely necessary to dissolve the chemical, the volume used
should not exceed the minimum volume necessary to dissolve or suspend
the chemical in the test solution. The upper limit of carrier volume is
0.5 mL/L and the same amount of carrier should be added to each con-
centration (refer to paragraph (g)(l) of this guideline).
(3) Test parameters, (i) The test temperature is to be 24 °C for
Selenastrum, Navicula, and Anabaena, and 20 °C for Skeletonema. Excur-
sions from the test temperature should be no greater than ±2 °C. Tempera-
ture should be recorded hourly during the test. A continuous recording
device is suitable for this purpose.
(ii) Test chambers containing Selenastrum, Navicula, and Anabaena,
must be illuminated continuously; those containing Skeletonema are to be
provided a 14-h light/10-h dark photoperiod. Fluorescent lights providing
4.3 K Ix (4,306 lm/m2 or 400+10% fc) for Selenastrum, Skeletonema,
and Navicula, and 2.2 K Ix for Anabaena are to be used. These lamps
should have a photo synthetically active radiation of approximately
66.5+ 10% (iEin/m2/sec. Light intensities (or light fluence rate) should be
measured at each test chamber position at the approximate level of the
test solution.
(iii) Stock algal cultures should be shaken on a rotary shaking appara-
tus. Test containers also should be placed on a rotary shaking apparatus
and oscillated at approximately 100 cycles/min for Selenastrum and at ap-
proximately 60 cycles/min for Skeletonema during the test. If clumping
of Skeletonema is experienced or anticipated, hand shaking once or twice
a day is acceptable. The rate of oscillation should be determined at the
beginning of the test or at least once daily during testing if the shaking
rate is changed or changes.
(iv) The pH of nutrient medium in which algae are subcultured is
to be 7.5 (+0.1) for Selenastrum, 8.1 (+0.1) for Skeletonema, 7.5 (+0.1)
for Navicula, and 7.5 (+0.1) for Anabaena, and is not adjusted after the
addition of the algae. The pH of all test solutions is to be measured at
the beginning and end of the test. If the test chemical is highly acidic
and reduces the pH of the test solution below 5.0 at the first measurement,
appropriate adjustments to pH should be considered, and the test solution
measured for pH on each day of the test.
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(v) Light intensity should be monitored at the beginning of the test
at the level of the test solution or at each test chamber position. If it is
suspected that light intensity has changed, monitoring more often during
the test will be necessary.
(f) Reporting. The sponsor must submit to the EPA all data devel-
oped by the test including those that are suggestive or predictive of acute
phytotoxicity. In addition to the reporting requirements as specified under
EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J,
the following specific information is to be reported:
(1) Detailed information about the test organisms, including the sci-
entific name, method of verification, strain, and source.
(2) Control charts of growth in the nontreatment and solvent controls
for each toxicity test.
(3) A description of the test chambers and containers, the volumes
of solution in the containers, the way the test was begun (e.g. conditioning,
test substance additions, etc.), the number of replicates, the temperature,
the lighting, and method of incubation, oscillation rates, and type of appa-
ratus. Specific modifications in test procedures due to using Anabaena or
Navicula must be noted.
(4) The concentration of the test chemical in the control and in each
treatment at the end of the test and the pH of the solutions.
(5) The number of algal cells per milliliter in each treatment and con-
trol and the method used to derive these values at the beginning, at 24,
48, and 72 h, and at the end of the test; the percentage of inhibition or
stimulation of growth relative to controls; and other adverse effect in the
control and in each treatment.
(6) The 96-h EC50 values, and when sufficient data have been gen-
erated, the 24-, 48-, and 72-h ECSOs and 95 percent confidence limits,
the methods used to derive these values, the data used to define the shape
of the concentration-response curve and the goodness-of-fit determination.
Electronic data submission (raw data) is encouraged to reduce data entry
time required to conduct statistical analyses.
(7) Methods and data records of all chemical analyses and test sub-
stance concentrations, including method validations and reagent blanks.
(8) The results of analyses such as: Microscopic appearance of algae,
size or color changes, percent mortality of cells and the fate of subcultured
cells, the concentration of test substance associated with algae and test
solution supernate or filtrate.
(9) If the range-finding test showed that the highest concentration of
the chemical tested (not less than 1,000 mg/L or saturation concentration)
8
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had no effect on the algae, report the results and concentration and a state-
ment that the chemical is of minimum phytotoxic concern.
(10) If the range-finding test showed greater than a 50 percent inhibi-
tion of algal growth at a test concentration at or below the analytical detec-
tion limit, report the results, concentration, and a statement that the chemi-
cal is phytotoxic at or below the analytical detection limit.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) American Society for Testing and Materials. ASTM E1218-20.
Standard guide for conducting 96-h toxicity tests with microalgae. In:
1991 Annual Book of ASTM Standards, Vol. 11.04: Pesticides; resource
recovery; hazardous substances and oil spill response; waste disposal; bio-
logical effects, pp 845-856 (1991).
(2) American Society for Testing and Materials. ASTM D 3978-80.
Standard practice for algal growth potential testing with Selenastrum
capricornutum. In: 1991 Annual Book of ASTM Standards, Vol. 11.04:
Pesticides; resource recovery; hazardous substances and oil spill response;
waste disposal; biological effects, pp 32-36 (1991).
(3) Payne, A.G. and R.H. Hall. A method for measuring algal toxicity
and its application to the safety assessment of new chemicals, pp 171-
180 in L.L. Marking and R.A. Kimerle (eds.). Aquatic Toxicology, ASTM
STM 667, American Society for Testing and Materials, Philadelphia, PA
(1979).
(4) Bruce, R.D. and D.J. Versteeg. A statistical procedure for model-
ing continuous toxicity data. Environmental Toxicology and Chemistry
11:1485-1494(1992).
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