&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention , '' ^:"'D
(7101) January 2012
Ecological Effects
Test Guidelines
OCSPP 850.3200:
Soil Microbial
Community Toxicity
Test
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NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number. Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also used by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this guidance, the Agency uses the word "should." In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.requlations.qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 850.3200: Soil microbial community toxicity test.
(a) Scope—
(1) Applicability. This guideline is intended to be used to help develop data to submit to
EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and
the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).
(2) Background. The source material used in developing this harmonized OCSPP test
guideline is the OPPT guideline under 40 CFR 797.3700 Soil Microbial Community
Toxicity Test. This guideline was formerly Public Draft OCSPP 850.5100 (April, 1996).
(b) Purpose. This guideline is intended for use in developing data on the toxicity of chemical
substances and mixtures ("test chemicals" or "test substances") subject to environmental effects
test regulations. The guideline prescribes a test using natural soil samples to develop data on the
toxicity of test substances to microbial populations indigenous to the soil. The Environmental
Protection Agency will use data from these tests to assess the hazard a test substance may present
in the environment.
(c) Definitions. The definitions in OCSPP 850.3000 apply to this guideline. The more specific
definitions in this paragraph also apply:
Ammonification is conversion of organic nitrogen compounds to ammonia (NHa) or to
ammonium ion (NH/i) compounds, performed by a variety of microorganisms in soil and
water.
Carbon dioxide (CO2) efflux is the evolution of CO2 gas from substrates mineralized by
microbial action, indicative of respiration.
kiloPascal (kPa) is a unit of pressure in the meter-kilogram-second system equivalent to
one newton per square meter (i.e., 1 Pa x 1,000) used as a measure of water availability in
soils.
Mineralization is the complete or ultimate degradation by microorganisms of organic
material to form inorganic end-products, e.g., carbon dioxide, water, chloride,
ammonium, nitrates, or orthophosphate.
Nitrification is the oxidation of ammonium salts to nitrites (NO2) and nitrates (NOs),
performed by relatively specialized microorganisms.
Surface soil is that layer of soil representing the top 15 centimeters (cm) of the area to be
sampled, excluding the litter horizon.
(d) General considerations—
(1) Summary of the test. Surface soil is sieved and supplemented with ground, dry
alfalfa. The test substance, if soluble, is added as a solution to moisten the soil, or is
added in a manner that best simulates its anticipated mode of entry in nature. All soil
samples are then incubated in darkness at approximately 22 degrees Celsius (°C). On
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days 5 and 28 after introduction of the test substance, samples are analyzed for NHs and
NOs content to establish ammonification and nitrification values, respectively, and for
CC>2 efflux as an indication of microbial respiration.
(2) General test guidance. The general guidance in OCSPP 850.3000 applies to this
guideline except as specifically noted herein.
(3) Range-finding test. A range-finding test is usually conducted to establish if
definitive testing is necessary and, if so, the concentrations of test substance to be used in
the definitive test. If the maximum concentration of test substance to which the soil
microbial community is likely to be exposed in nature can be predicted, soil samples
should be treated with concentrations that are 0.1, 1, and 10 times the anticipated
environmental exposure concentration. Should reasonable predictions of anticipated
environmental exposure concentrations not be possible, soil samples should be exposed
to a series of widely spaced concentrations of the test substance (e.g., 1, 10, 100, 1,000,
10,000 micrograms (jig) test substance per gram (g) of soil). The test should consist of
exposing at least two samples of soil from the same source to each concentration of test
substance and to each control. Two samples are the minimum number used for
measurement of test endpoints. The details of the test do not have to be the same as for
definitive testing. Treatment replication is not needed and nominal concentrations of the
chemical are acceptable. However, the range-finding test will be more useful the greater
the similarity between the range-finding test and the definitive test. Report results of
range-finding tests along with the results of the definitive test, if range-finding tests are
conducted.
(4) Definitive test. The purpose of the definitive test is to evaluate the toxicity of the test
substance to the community of microorganisms residing in a particular soil and to
delineate the concentration-response curve and ECso (with 95 percent (95%) confidence
intervals) for each of three variables (CO2 evolution and NHs and NOs soil content) that
indicate the capacity of the soil microbial community to decompose organic matter and
release plant nutrients. The slopes of the concentration-response curves, associated
standard errors, and the 95% confidence intervals of the slopes should also be
determined. For this determination, a minimum of five concentrations of the test
chemical, plus appropriate controls, are tested. For each soil source tested, the
concentration range should be selected to define, as closely as possible, the
concentration-response curve between the ECio and ECgo for each variable. Analytical
confirmation of the test concentrations is performed as described in OCSPP 850.3000.
(e) Test standards—
(1) Test substance. The substance to be tested should be technical grade, unless the test
is designed to test a specific formulation, mixture, or end-use product. OCSPP 850.3000
lists the type of information that should be known about the test substance before testing,
and discusses methods for preparation of test substances.
(2) Test duration. The test duration is 28 days after application of the test substance.
(3) Test organism. No particular species of test organisms are recommended for use in
this test due to the emphasis placed on maintaining the natural state of the soil samples
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and their resident populations of microorganisms. The test organisms are those that occur
naturally in the soil; no other test organisms are introduced into the system. Within a
given test, all soil, including that of the controls is from the same source.
(4) Administration of test substance. Depending upon the properties of the test
substance, it is added via a stock solution (aqueous or solvent vehicle) to the soil,
adsorbed onto the soil, or directly mixed into the soil.
(i) Preparation methods.
(A) Reagent water should be used in making stock solutions of a water-
soluble test substance. Sufficient quantities of each concentration should
be available to minimize storage time and disposal volume. A constant
volume of the stock solution should be added at the beginning of the test
to each soil sample designed to receive the test substance. OCSPP
850.3000 contains additional information on the preparation of stock
solutions.
(B) A test substance that is insoluble in water, but which can be suspended
in an aqueous solution by a vehicle, should be added, with the vehicle, to
those soil samples designated to receive the test substance. The vehicle
should be soluble in water, nontoxic to microbial life at the concentration
applied, and used in a minimal amount to dissolve or suspend the test
substance. There are no preferred vehicles; however, acetone, gum arable,
ethanol, and others have been used extensively in testing herbicides, plant
growth regulators, fungicides, and other chemical substances that affect
plants. Any such vehicle may be used for this test, providing it neither
enhances nor inhibits the activities of the soil microbes. Vehicle controls
are included in the experimental design and tested simultaneously with the
test substance, if a vehicle is used. OCSPP 850.3000 contains additional
information on preparation of stock solutions using vehicles.
(C) A water-insoluble test substance for which no nontoxic, water-soluble
vehicle is available should be dissolved in an appropriate volatile solvent.
The solution should be mixed with the ground alfalfa soil supplement,
then placed in a rotary vacuum apparatus and evaporated, leaving a
uniform coating of the test substance on the alfalfa. A portion of the
alfalfa is then weighed; the test substance extracted with the same organic
solvent, and then the test substance is assayed before the alfalfa is mixed
with the soil in the test containers. Solvent controls (i.e.., alfalfa treated
only with solvent) should be included in the experimental design and
tested simultaneously with the test substance.
(D) If the test substance is not readily soluble in water or in another
commonly used vehicle, and is known to be applied or transported in
nature directly to the soil as a previously prepared liquid or powder, it
should be mixed, in its liquid or dry form, directly into the soil samples.
To ensure equal distribution of the test substance throughout each test
sample, mix thoroughly.
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(ii) Test concentrations. For this determination, a minimum of five treatment
levels of the test chemical, plus appropriate controls, are tested.
(iii) Soil. To be appropriate for this guideline, a soil should possess a pH of 4 to
8, organic matter content between 1% and 8%, a cation exchange capacity (CEC)
greater than 7 milliequivalents per 100 grams, and consist of less than 70% sand.
Soils to which fertilizer or pesticide(s) have been applied within the past 24
months should be avoided. Soil collections should be restricted to the surface
soil. Large objects should be removed manually, and the remaining soil allowed
to air-dry until sievable (approximately 12% water content), after which it is
passed through a 2-millimeter (mm) mesh screen. For each sample, an amount of
soil equivalent to approximately 50 grams (g) oven-dry weight should be placed
in an inert container. At least one of these samples, considered to provide a
baseline measurement, should be extracted immediately to determine NHs and
NOs content (see paragraph (d)(4) of this guideline). Alfalfa, dried and ground to
pass through a 0.6-mm mesh screen should be added (0.3 g) to each remaining
sample and the sample should be thoroughly mixed. Water content of the soil
should be adjusted to approximately 10 kPa by adding reagent (distilled,
deionized or reverse osmosis) water containing the desired concentration of test
substance (in vehicle, if necessary). If insoluble in both water and commonly
used vehicles, the test substance should be mixed into the soil as a solid and the
appropriate amount of water added subsequently. The test containers may be
covered with 0.13 jim (0.5 mil) polyethylene to minimize water loss, yet permit
gas exchange, or left open and watered to their original weight every 7 days.
Regardless, the test substance should be applied only during the original watering.
(5) Controls.
(i) Every test includes a negative control, and a vehicle (solvent) control if a
vehicle is used. Controls consist of the same soil, soil supplements, test
conditions, and procedures, except that no test substance is added.
(ii) If an aqueous stock solution was used to prepare test concentrations, controls
should receive an equal volume of reagent water without the test substance. If a
vehicle was used to dissolve or suspend the test substance, a vehicle control (i.e..,
solvent in water without the test substance) should also be included. Should the
test substance be a powder that is mixed directly into soil and subsequently
moistened with distilled water; the control should receive an equal volume of such
water only.
(6) Number of replicates. A total of 10 replicates of each test substance concentration,
control, and vehicle control (if applicable) are used. This provides two sets of five
replicates, one set for the analyses conducted 5 days after test substance application and
one set for the analyses conducted 28 days after test substance application. The
assignment of soil containers to test substance concentrations should be random. In
addition, placement of the containers in the incubation chamber should be randomized.
(7) Facilities, apparatus and supplies—
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(i) Environmental chambers or incubators. Environmental chambers or
incubators should provide adequate space and controls necessary to incubate
numerous soil samples in total darkness at a constant temperature for prolonged
periods of time. Chambers should be designed to prevent escape of internal air
into the external environment other than through appropriate filtering material or
media to prevent contamination of the external environment with the test
substance.
(ii) Test containers. For each test, at least 60 to 70 soil containers (two series of
five per concentration of test substance, two series of five for the control, and two
series of five if a vehicle control is necessary) should be used. In addition, soil to
be extracted immediately as the control is most easily handled in an identical
container. All containers used in each experiment should be of equal size and
volume, posses the same configuration, and should be made of the same inert
material. Wide mouth jars (for example, glass canning, 0.5 pint or 110-millilitre
(mL) capacity) are adequate for this purpose. Test containers may be covered
with polyethylene film (0.13 micrometer (|im); 0.5 mil) to prevent water loss.
Control containers should be handled identically to the test containers except that
none of the test substance is added.
(iii) Cleaning and sterilization.
(A) Soil containers and test solution storage containers should be cleaned
before use. All equipment should be washed to remove any residues
remaining from manufacturing or prior use. A dichromate solution should
not be used for cleaning containers.
(B) If cleaning and rinsing of previously used soil containers has been
thorough, the effects of any microorganisms remaining on the interior
surface of the containers should be insignificant in the presence of the new
test soil. Sterilization should not be necessary, but is considered an
acceptable option.
(C) Soil treated with the test substance and solvent control soil should be
discarded at the end of the experiment. Disposal should conform to
applicable regulations.
(8) Environmental conditions. Environmental conditions should be maintained as
follows:
(i) Temperature. The temperature should be 22 °C (or that temperature to which
the microorganisms are most accustomed in nature) and it should be constant
within ± 2°C during the test.
(ii) Lighting and photoperiod. Total darkness should be used during incubation
to prevent photosynthesis by algae or the growth of moss.
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(9) Observations-
(i) Measurement of test substance. Analytical confirmation of the concentration
of test substance in the stock solutions and/or in the soil should be performed as
described in OCSPP 850.3000. Validated analytical methods are used to measure
the amount of test substance in a sample before beginning the test, as described in
OCSPP 850.3000.
(ii) Environmental conditions—
(A) Temperature. Temperature in the incubation chamber should be
monitored continuously.
(B) Stock solution pH. The pH of the stock solutions should be measured
before use.
(iii) Measures of effect—
(A) Soil ammonia, nitrate and nitrogen content and carbon dioxide
efflux. Of the soil samples prepared for each test concentration or control,
one set of five replicate samples should be assayed after 5 days of
exposure to the test substance for NHs and NOs content, and then
discarded. A second set of 5 replicate samples should be analyzed the
same day for CO2 evolution and then reincubated (dark, 22 °C). On day
28 after exposure to the test substance, all remaining (reincubated)
samples should be assayed first for CO2 efflux and then again for nitrogen
content.
(B) Microorganisms. On days 5 and 28 after introduction of the test
substance, the effects of treatment should be assessed as the CO2 efflux
rate and the NOs and NHs concentrations per gram of dry soil in treated
samples, relative to untreated controls and, if applicable, vehicle controls,
and to values in freshly sieved (pretreatment) soil.
(1) To measure CO2 efflux, each test container is closed with a
two-hole stopper fitted with Teflon tubes and twistcock connectors
for attachment to the measurement apparatus. The apparatus (see
Figure 1) should deliver a stream of humid, CO2-free air to the test
system, and the effluent air should be dried, diluted, and delivered
for infrared gas analysis (IRGA). The period of incubation should
be adjusted to match the CO2 efflux rate with the detection
capability of the IRGA, and may vary from 1 to 77 hours. In lieu
of IRGA, CO2 may be trapped in a hydroxide solution and titrated,
or measured by gas chromatography. CO2 efflux rates are
expressed in terms of micrograms per gram of dry soil per hour.
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Figure 1.—Flow Diagram for the IRGA Method of Determining CO2 Evolution from
Soil Samples
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(2) Accumulation of inorganic nitrogen is measured by extracting
each soil sample with 80 mL of 1 normal potassium chloride (N
KC1) solution. After adding KC1 and shaking each container by
hand to suspend the soil, sample containers should be placed on a
rotary shaker at high speed for 1 hour, then shaken again by hand
to resuspend the soil. Samples should be filtered (Whatman 42
low-nitrate filter paper) and the extract (filtrate) should be
analyzed for NOs and NHs. EPA or ASTM methods should be
used for these analyses. NOs and NH4 concentrations are
expressed in terms of micrograms per gram of dry soil.
(f) Treatment of results—
(1) Data summary—
(i) Environmental conditions. Temperature data should be summarized in
tabular form, showing the mean, standard deviation, and range of temperature
during the test.
(ii) Carbon dioxide efflux rates, and soil nitrate and ammonia content. CC>2
efflux rates and NOs and NH3 concentrations in treated samples and from
untreated controls, aqueous or solvent vehicle controls (if a vehicle is used), and
from the freshly sieved, pretreatment soil should be summarized in tabular form
by observation date. Means and standard deviations should be plotted for each
treated sample and each control.
(2) NOEC. The significance of differences between means may be established using
analysis of variance and a multiple range test such as Dunnett's. These hypothesis-
testing procedures can be used to determine NOEC and LOEC values based upon the
CC>2 efflux, nitrification and ammonification.
(3) Concentration-response curve, slope, and ECso- The ECso for each of CO2 efflux,
nitrification and ammonification should be determined. Appropriate statistical analyses
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should provide a goodness-of-fit determination for the concentration-response curves.
Standard error and 95% confidence intervals for the calculated ECso values are to be
included. The slope of the concentration-response curve, its standard error, and 95%
confidence interval should also be reported.
(4) Statistical methods. Statistical procedures for modeling continuous data are
available and should be used. Additional discussion about endpoints and statistical
procedures is found in OCSPP 850.3000.
(g) Tabular summary of test conditions. Table 1 lists the important conditions that should
prevail during the definitive test. Meeting these test conditions will greatly increase the
likelihood that the completed test will be acceptable or valid.
Table 1.—Summary of Test Conditions for Soil Microbial Community Toxicity Test
Test type
Test duration
Substrate
Temperature
Lighting
Test container size
Number of replicate containers per test treatment
Test treatment levels
Test substance application method
Measures of effect (Measurement endpoints)
Spiked natural soil
28 days
Soil, < 70% sand, pH of 4 - 8, organic content 1 -
8%, CEC > 7 meq/100 g; sieved to 2 mm
22 °C (constant during test within ± 2 °C)
Complete darkness at all times
Sufficient to contain 50 g soil (e.g., 0.5 pint jars)
10 (minimum)
Minimum of 5 treatment levels plus appropriate
controls
Via aqueous stock solution, vehicle stock solution,
adsorbed to the alfalfa supplement, or directly
mixed into the soil.
EC50 and NOEC/LOEC based upon CO2 efflux,
ammonification and nitrification at day 5 and 28
(h) Test validity. This test would be considered to be unacceptable or invalid if one or more of
the conditions in Table 2 occurred. This list should not be misconstrued as limiting the reason(s)
that a test could be found unacceptable or invalid. However, except for the conditions listed in
Table 2 and in OCSPP 850.3000, it is unlikely a study will be rejected when there are slight
variations from guideline environmental conditions and study design unless the control
organisms are significantly affected, the precision of the test is reduced, the power of a test to
detect differences is reduced, and/or significant biases are introduced in defining the magnitude
of effect on measurement endpoints as compared to guideline conditions. Before departing
significantly from this guideline, the investigator should contact the Agency to discuss the reason
for the departure and the effect the change(s) will have on test acceptability. In the test report, all
departures from the guideline should be identified, reasons for these changes given, and their
effects on test endpoints noted and discussed.
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Table 2.—Test validity elements for the soil microbial community toxicity test
1. All test containers were not identical and did not contain the same amount of soil from the same
source.
2. Untreated (negative controls) and appropriate solvent controls were not included in a test.
(i) Reporting—
(1) Background information. Background information to be supplied in the report
consists at a minimum of those background information items listed in paragraph (j)0) of
OCSPP 850.3000.
(2) Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences which
may have influenced the results of the test.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, and its purity (i.e. for pesticides, the identity and
concentration of active ingredient(s)). If radiolabeled substance was used provide
the radio purity and location(s) of the label.
(ii) Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance in the range-finding and
definitive test: description of test chemical introduction into the test medium (e.g.,
directly mixed, as an aqueous or solvent stock solution, or adsorbed onto the
alfalfa supplement), the mass and volume of soil and test substance used for each
treatment, how test substance was mixed into the soil.
(iv) For the definitive test, the number of treatments and nominal concentration at
test initiation of the test substance per unit dry weight of test soil when the test
substance is introduced as dissolved in water, solubilized with a vehicle, or coated
on the alfalfa supplement and/or mixed directly into the soil.
(v) Description of stock solution preparation: the name and source of the vehicle,
the nominal concentration(s) of the test substance in the vehicle, in stock
solutions, or mixtures, and the vehicle concentrations used in the treatments and
controls, pH of test solutions applied to the soil samples.
(4) Soil microorganisms.
(i) Source of the test soil, the type of ecosystem from which it was removed, its
chemical and physical characteristics (mechanical analysis), and any available
geological information including soil type (classification).
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(ii) Collection method, storage, and handling of test soil prior to introduction of
test substance.
(iii) Source of alfalfa and amount added to soil.
(5) Test system and conditions. Description of the test system and conditions used in
the definitive test and any preliminary range-finding tests.
(i) Description of the test containers: type, size, volume, material, and
conditioning method.
(ii) Description of incubation chamber: type and size.
(iii) Volume and weight of soil used in each test container.
(iv) Number of replicates per treatment level and control group(s).
(v) Frequency and methods of adding water to soil containers during the test
period.
(vi) Description of experimental design and/or arrangement of equipment,
including a diagram, if complex.
(vii) Methods used to determine the placement of soil containers in the incubation
chamber and the assignment of test concentrations to containers to ensure
randomization of exposure.
(viii) Test duration.
(ix) Methods and frequency of environmental monitoring performed during the
definitive test for temperature and humidity.
(x) Method, type and frequency of observations made on soil microorganisms
during the test.
(xi) For the definitive test, all analytical procedures and preservation methods
should be described. The accuracy of the method, method detection limit, and
limit of quantification should be given.
(6) Results.
(i) Environmental monitoring data results (temperature and humidity) in tabular
form (provide raw data for measurements not made on a continuous basis), and
descriptive statistics (mean, standard deviation, minimum, maximum).
(ii) Micrograms of CC>2 evolved per gram of dry soil per hour, and micrograms of
each of NH3 and NOs present per gram of dry soil, by soil source and treated and
untreated samples at day 5 and 28 in tabular form (provide raw data).
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(iii) For the definitive test, report the 5-day and 28-day ECso values, with 95%
confidence intervals for ammonification, nitrification, and CC>2 evolution. The
slopes of the concentration-response curves, associated standard errors, and the
95% confidence intervals should be reported as well.
(iv) Description of statistical method(s) used, including software package, for
determining ECso, LOEC and NOEC, values and the concentration-response
model parameters and the basis for the choice of method. Provide results of any
goodness-of-fit tests and determination of minimum significant differences.
(j) References. The references in this paragraph should be consulted for additional background
material on this test guideline.
(1) Hammons, A.S. (Ed.), 1981. Methods for ecological toxicology. A critical review of
laboratory multispecies tests. ORNL-5708. EPA-560/11-80-026, Office of Toxic
Substances, U.S. EPA, Washington, DC and Oak Ridge National Laboratory, Oak Ridge,
TN.
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