United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-015

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.3200:

Soil Microbial 

Community Toxicity 

Test
i

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.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
Page 1 of 11

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 (NH3) or to 

ammonium ion (NH4) 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 (NO3), 

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 NH3 and 

NO3 content to establish ammonification and nitrification values, respectively, and for 

CO2 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 (μg) 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 EC50 (with 95 percent (95%) confidence 

intervals) for each of three variables (CO2 evolution and NH3 and NO3 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 EC10 and EC90 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 
Page 3 of 11

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 watersoluble 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 arabic, 

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.
Page 4 of 11

(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 NH3 and 

NO3 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 μm (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—
Page 5 of 11

(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 (μm); 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 NH3 and NO3 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 NO3 and NH3 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

(2) Accumulation of inorganic nitrogen is measured by extracting 

each soil sample with 80 mL of 1 normal potassium chloride (N 

KCl) solution. After adding KCl 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 NO3 and NH3. EPA or ASTM methods should be 

used for these analyses. NO3 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. CO2

efflux rates and NO3 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 hypothesistesting procedures can be used to determine NOEC and LOEC values based upon the 

CO2 efflux, nitrification and ammonification. 

(3) Concentration-response curve, slope, and EC50. The EC50 for each of CO2 efflux, 

nitrification and ammonification should be determined. Appropriate statistical analyses 

WATER

CO2 FREE

WATER

TRAP SODA

LIME TRAP

REGULATOR

VALVE

AIR

PUMP

INFRARED

GAS

ANALYZER

MAGNETIC

STIRRER

DILUTION

JAR

FLOW METER

DRYING

AGENT

SOIL

SAMPLE

RELEASE VALVE
Page 8 of 11

should provide a goodness-of-fit determination for the concentration-response curves. 

Standard error and 95% confidence intervals for the calculated EC50 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 Spiked natural soil

Test duration 28 days

Substrate Soil, < 70% sand, pH of 4 - 8, organic content 1 -

8%, CEC > 7 meq/100 g; sieved to 2 mm

Temperature 22 °C (constant during test within ± 2 oC)

Lighting Complete darkness at all times

Test container size Sufficient to contain 50 g soil (e.g., 0.5 pint jars)

Number of replicate containers per test treatment 10 (minimum) 

Test treatment levels Minimum of 5 treatment levels plus appropriate 

controls

Test substance application method Via aqueous stock solution, vehicle stock solution, 

adsorbed to the alfalfa supplement, or directly 

mixed into the soil. 

Measures of effect (Measurement endpoints) 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)(1) 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 CO2 evolved per gram of dry soil per hour, and micrograms of 

each of NH3 and NO3 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). 
Page 11 of 11

(iii) For the definitive test, report the 5-day and 28-day EC50 values, with 95% 

confidence intervals for ammonification, nitrification, and CO2 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 EC50, 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.