United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-003

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.4900:

Terrestrial Soil-Core 

Microcosm 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.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.
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OCSPP 850.4900: Terrestrial soil-core microcosm 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 797.3775 Soil-Core Microcosm.

(b) Purpose. This guideline is intended for use in developing data on the toxicity and fate of 

chemical substances and mixtures (“test chemicals” or “test substances”) subject to 

environmental effects test regulations. This guideline describes a soil-core microcosm test, 

which provides information on the potential fate and ecological effects of chemical substances 

released to a specific terrestrial ecosystem. The Environmental Protection Agency (EPA) will 

use data from this test in assessing the hazard or risks a test substance may present in the 

terrestrial environment.

(c) Definitions. The definitions in the OCSPP 850.4000 guideline apply to this test guideline. 

In addition, the definitions in this paragraph also apply:

Bioconcentration factor (BCF) refers to, in this guideline, the ratio of the concentration 

of test substance in plant tissue (i.e., biota) to that in soil.

Biota refers to, specifically in this guideline, the organisms in the soil at the time of 

extraction of the core and the natural vegetation or crop species introduced as the 

autotrophic component. Biota includes all heterotrophic and carnivorous invertebrates in 

the soil and all soil and plant bacteria, fungi, and viruses.

Reagent water refers to water that has been prepared by deionization, glass distillation, or 

reverse osmosis (see the OCSPP 850.4000 guideline).

Soil-core refers to an intact, undisturbed (non-homogenized) core of soil, that is extracted 

in situ from a soil type typical of the region or site of interest, and that is of sufficient 

depth to allow a full growing season for the natural vegetation or the crop(s) selected, 

without causing the plants to become significantly root bound.

Soil-core microcosm refers to a physical miniaturized model of an interacting community

of soil autotrophs, omnivores, herbivores, carnivores, and decomposers within an intact 

soil profile.

(d) General considerations―

(1) Summary of the test. The purpose of the soil-core microcosm test is to determine 

the potential fate and ecological effects of a test substance, including its transformation 

products, released to a specific terrestrial soil-plant ecosystem. A soil core, as shown in 

Figure 1, containing biota (soil and plants or crop(s)) typical of the region of interest, is 
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treated with the test substance and maintained under controlled conditions in either a 

growth chamber or greenhouse. The test is usually conducted for a minimum of 12 

weeks, from first application of the test substance to final harvest. Single or multiple 

applications of the test substance may be chosen, depending on the expected mode of 

introduction of the test substance into the environment. Leachate, soil, and plant samples 

are analyzed to evaluate the environmental fate of the test substance. Ecological effects 

of the test substance are evaluated on the basis of measurements of primary productivity 

and nutrient loss, as well as on determinations of BCFs and observations of plant 

condition. Chemical substances with high vapor pressures or high Henry's law constants 

should not be tested in the soil-core microcosm as described in this guideline.

Figure 1.—Microcosm Structure and Materials

(2) General test guidance. The general guidance in the OCSPP.4000 guidance applies 

to this guideline except as specifically noted herein.

(3) Range-finding test. A range-finding test may be conducted to determine the 

concentrations of test substance to be used in the definitive test. In the range-finding test, 
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the microcosms are treated at a series of widely spaced concentrations of the test 

substance. Microcosms should be treated with a minimum of five concentrations of the 

test substance. Concentrations typically used for treatment are 0.1, 1.0, 10, 100, and 

1,000 micrograms per gram (μg/g) in the upper 20 centimeters (cm) of topsoil of the 

microcosm, if actual environmental concentrations are not known and cannot be 

predicted. The bulk density in grams per cubic centimeter (g/cm3) of the dry soil should 

be used to calculate the exposures. Depending on the expected mode of release of the 

test substance, each recommended concentration may be applied as a single dose or may 

be divided into multiple doses. In either case, the final amount of test substance applied 

for any given treatment level should add up to the recommended total concentration for 

that treatment level (e.g., 4 multiple doses of 0.25 μg/g which totals 1 μg/g or a single 

dose of 1 μg/g). Treatments do not need to be replicated and nominal concentrations of 

the test substance are acceptable.

(i) Physicochemical information supplied for the test substance should be used to 

tailor the general range-finding test procedures to the specific substance.

(ii) Phytotoxicity and/or bacteriostatic action, if known, should be considered in 

selecting exposure concentrations for the range-finding test. Only one 

concentration greater than that known to cause at least a 50 percent (50%) change 

in plant growth or a 50% change in bacterial growth/respiration should be tested.

(iii) The range-finding test should last at least 4 weeks from first application of 

the test substance to plant harvest.

(iv) At the termination of the range-finding test, soil samples should be collected 

from the top, middle, and bottom of the 60-cm soil cores. If the radiolabeled test 

substance or its transformation products are not detected in the deeper soil 

samples by liquid scintillation counting, soil samples at the end of the definitive 

test should be taken nearer the top of the soil column.

(v) If no discernible effects (e.g., plant growth, soil organism 

diversity/abundances, BCF, nutrient loss (see paragraph (d)(1) of this guideline)) 

of the test substance are detected during the range-finding test at one-half of 

saturation or 1,000 μg/g (whichever is higher), including visible effects of plant 

injury, no definitive test is necessary. 

(4) Definitive test. The purpose of the definitive test is to determine the potential fate 

and ecological effects of a test substance, including its transformation products, in a sitespecific natural grassland or agricultural ecosystem. The test substance is introduced into 

the test system in a manner representative of the expected mode of entry into the 

environment. The fate or final distribution of the test substance and its transformation 

products are determined by methods appropriate to the test, including sensitivity factors 

adequate to verify exposure and distinguish between the test substance, its transformation 

products, and naturally occurring materials present in the test system. Whenever 

possible, this involves use of a radiolabeled test substance, and subsequent analysis of the 
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primary microcosm compartments and soil leachate for radioactivity and chemical 

identity. The elements of an acceptable definitive test are given in Table 1.

(e) Test standards―

(1) Test substance.

(i) The form of the test substance used should approximate a reasonable scenario 

of how the substance is expected to be released into the environment. Water 

solubility and dissociation constant(s) of the test substance and soil pH must be 

considered in determining the formulation of the test substance.

(ii) Whenever possible, the test substance should be radiolabeled. The label may

be stable isotopes such as carbon-14 (

14C), nitrogen-15 (15N), or other suitable 

labels and, if possible, should be located in a portion (or portions) of the molecule 

known or expected to persist and/or have biological activity. For single chemical 

substances, two or more portions of the molecule may need to be labeled; in the 

case of mixtures, for a satisfactory test each component should be labeled and 

studied separately.

(2) Test duration. The test duration is typically 12 weeks from the first application of 

the test substance to the final harvest of test organisms. The test may be extended beyond 

12 weeks to accommodate plant species which take longer to reach the desired maturity 

(e.g., seed production).

(3) Test microcosm―

(i) Soil cores. Soil cores (17-cm diameter by 60-cm deep) should be extracted 

from either a natural grassland ecosystem or a typical agricultural soil in the 

region of interest. The intact system should be extracted with a specially 

designed, steel extraction tube, as shown in Figure 2, and a backhoe. 

Disturbances during extraction and preparation of the soil core should be 

minimized.

(ii) Agricultural microcosm―

(A) Soil-core. For an agricultural microcosm, the soil which is plowed 

(generally, the top 15 cm of soil) should be moved aside and saved. Once 

the core is cut by the leading edge of the driving tube, it should be forced 

up into the microcosm tube or Driscopipe® (high density, high molecular 

weight polyethylene pipe) as demonstrated in Figure 1 in this guideline. 

The polyethylene pipe should then contain a 45-cm core of subsoil. The 

microcosm tube is placed on a Buchner funnel covered by a thin layer of 

glass wool and the homogenized topsoil that was saved should be 

backfilled into the upper 15 cm of the microcosm tube after it has been 

returned to the laboratory.
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(B) Test plant species. 

(1) A mixture of grasses and broad leaves (e.g., legumes) are added 

to the soil-core in the laboratory. Seeds from two or three species 

of grasses or legumes that are typically grown together as an

agricultural crop in the region of interest should be chosen and 

planted. Chosen species should be compatible and able to grow to 

maturity in the limited surface area of the microcosm. Thus, large 

crops such as corn or sorghum cannot be used under these

guidelines. The rate of seed application should duplicate standard 

farming practice for the region of interest. Seeds should be planted 

evenly and covered to an appropriate depth with soil.

(2) Information on seed lot, seed year, or growing season collected 

and germination percentage should be provided by the source of 

the seed. Only untreated seed (not treated with fungicides, 

repellants, etc.) taken from the same lot and year or season of 

collection should be used in a given test. In addition, all seed of a 

species used in a test should be of the same size class, and that size 

class which contains the most seed should be selected and used in a 

given test. Any damaged seed should be discarded.

(iii) Natural grassland microcosm―

(A) Test plant species. For a natural grassland microcosm, the vegetation 

covering the natural grassland ecosystem should be clipped to a uniform 

height before the core is extracted. Natural plant cover should be 

sufficiently diverse to be representative of plant species in the region of 

interest.
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Figure 2.—Diagram of Microcosm Extraction Tube

(B) Soil core. The soil core from the grassland ecosystem should be 

removed as a single unit (soil and polyethylene pipe) from the extraction 

tube, taken to the laboratory, and placed on a Buchner funnel covered by a 

thin layer of glass wool.

(iv) Leachate collection. Data regarding solubility of the test substance in water 

and its capacity to sorb to soils should be used, along with the results of the rangefinding test, to help determine the appropriate regime for soil leachate collection 

and analysis. Microcosms should be leached, as described in paragraph 

(e)(9)(iii)(C) of this guideline, at least twice before application of the test 

substance and once every 2 or 3 weeks after such application. The frequency of 

rainfall in the region of interest should be considered when a leaching regime is 

selected. Water to leach the microcosm should be added to each microcosm over 

an 8- to 12-h period to avoid waterlogging the soil surface. To ensure that all test 

microcosms will leach within a 2-day period, at least 15% more soil cores should 

be extracted than are required for the tests. When the microcosms are leached 

before planting, those which do not leach, leach too quickly, or take longer than 2 
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days to produce 100 mL of leachate after the soil has been brought to field 

capacity, should be discarded.

(4) Administration of test substance―

(i) Exposure methods. The method and pattern of application and the form of 

the test substance used should approximate a reasonable scenario of how the 

substance is expected to be released into the environment. The method and 

pattern of application should also reflect the actual or predicted field situation. 

Single or multiple applications may be used.

(A) For compounds likely to be found or partition to rainfall or leachate, 

in a simulation of a "realistic" exposure scenario the primary mode of 

exposure to the test substance is anticipated to be by addition of pHadjusted, reagent water or rainwater containing appropriate concentrations 

of the test substance. In these cases, the procedure in paragraphs 

(e)(4)(i)(A)(1) through (e)(4)(i)(A)(4) of this guideline is recommended.

(1) Test substances which are likely to be released into the 

environment as a liquid or powder, and which can be mixed with 

water, should be applied as a single dose of liquid in a volume 

sufficient to bring the A horizon of the soil surface of the 

microcosm to field capacity.

(2) Water simulating rainfall or leaching should be filtered 

rainwater from the site being evaluated or reagent water with a 

known chemical composition.

(3) The volume of reagent water or rainwater used for laboratory 

microcosms may be determined on-site using a microcosm of the 

same soil type without vegetation. The volume selected should be 

identical for all microcosms and be sufficient to bring the A 

horizon of the soil surface to field capacity.

(4) Carriers other than water should not be used unless they are 

likely to be released into the environment with the test substance. 

If a vehicle is necessary, acetone or ethanol should be considered; 

however, the use of vehicles should be avoided unless they are 

essential to produce a realistic exposure.

(B) Several typical methods of application are suggested for particular 

types of test substances:

(1) Irrigation water. If the test substance is likely to be a 

contaminant of irrigation water, it should be applied periodically,

such as daily or weekly, in proportionate concentrations, such that 

the total amount applied equals the desired level of treatment.
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(2) Sprayed on growing plants. If the test substance is normally 

sprayed on growing plants, the desired amount should be mixed 

with the volume of water necessary to wet the soil surface and wet 

the plants to the point at which they begin to drip. A

chromatography sprayer should be used to spray plants that are 

past the seedling stage. The manufacturer's recommendations for 

field spraying the test substance should be followed as closely as 

possible and the test run at least 8 weeks after plants are sprayed.

(3) Aerosol or powder on growing plants. If the test substance is 

applied as an aerosol or powder, plants should be sprinkled 

immediately after treatment to avoid resuspension of particulates 

and reduce the potential for cross-contamination of exposure 

concentrations.

(4) Low water solubility test substance. If the test substance does 

not mix with water, it should be applied as evenly as possible to 

the top of the microcosm. If the microcosm is simulating an 

agricultural system, the test substance should be mixed into the 

topsoil before planting.

(ii) Treatment levels. At least three treatment levels of the test substance are 

tested. The treatment concentrations should be selected to produce a 20 to 25% 

change in plant productivity in each treatment based on results from the rangefinding test. The treatment concentrations should bracket the known or expected 

environmental concentration of the test substance. However, if the environmental 

concentration is unknown and cannot be estimated, the maximum concentration 

of the substance in solution should not exceed half of saturation.

(5) Controls. Every test includes controls consisting of the same soil core material, test 

conditions, procedures and test population except that no test substance is added. In 

addition vehicle (solvent) controls are also included if a vehicle is used.

(6) Number of replicate microcosms. 

(i) At a minimum ten replicate microcosms are used for each of the three 

concentrations and for the control, for a total of 40 microcosms. The 10 replicate 

microcosms in each treatment group should be used as five replicate pairs. Six 

microcosms are typically contained in a moveable cart which is packed with 

styrofoam beads, as shown in Figure 3 of this guideline. Microcosms that have 

been paired for analysis should be placed in different carts to ensure that 

environmental conditions are as uniform as possible.

(ii) An appropriate random process should be used, such as completely

randomized, randomized block, or Latin-square design, to assign microcosms to 

different concentrations of the test substance. To minimize location-induced 

effects, which can have a significant impact upon plant growth, the placement of 
Page 9 of 22

each microcosm cart should be moved once per 7 days in the greenhouse or 

growth room to minimize.

(7) Facilities, apparatus and supplies―

(i) Greenhouse or growth room. The greenhouse or growth room should 

provide adequate environmental controls to meet light and temperature 

specifications.

(ii) Analytical laboratory facilities and disposal facilities. Laboratory facilities 

for test substance determinations should include: Nonporous floor covering; 

absorbent bench covering with nonporous backing; and adequate disposal 

facilities to accommodate radiolabeled test solutions and wash solutions 

containing the test substance at the end of each test, and any bench covering, 

laboratory clothing, or other contaminated materials; appropriate equipment for 

analytical determinations, drying ovens, refrigerators, and standard laboratory 

glassware.

(iii) Core extraction equipment. A specially designed steel extraction tube and 

a backhoe are needed to extract soil cores.

(iv) Containers and support equipment. 

(A) The three basic materials used for a single microcosm are: a 60-cm 

long Driscopipe® tube (17.5 cm diameter), a 186 millimeter (mm)-

diameter porcelain Buchner funnel, and a thin layer of glass wool (see 

Figure 1 of this guideline). Containers used in each test should be of equal 

size and volume and possess the same configuration.

(B) Several mobile carts should be used to hold the microcosms. The carts 

should be designed to hold adequate styrofoam beads for insulation, as 

shown in Figure 3 of this guideline. 
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Figure 3.—Arrangement of Microcosms in Styrofoam Cart

(v) Cleaning. All equipment used in the test should be cleaned before use and 

should be washed according to good standard laboratory practices, to remove any 

residues remaining from manufacture or use. The funnel and tube should be 

washed with acid (50% concentrated nitric acid (HNO3)) before use and then 

rinsed with reagent water. A dichromate solution should not be used for cleaning 

containers.

(8) Environmental conditions. Microcosms should be kept in a greenhouse or 

environmental chamber with controlled environmental conditions.

(i) Temperature. The temperature should approximate outdoor temperatures that 

occur during a typical growing season in the region of interest.

(ii) Lighting and photoperiod. The photoperiod and intensity of light typical for 

the growing season in the region of interest should be simulated. Light for the test 

system can be supplied by artificial lighting suitable for plant growth in either an 

environmental chamber or greenhouse or can be the natural photoperiod occurring 

in a greenhouse. If the test is performed in an environmental chamber, the daily 

photoperiod for the microcosm should be at least the average monthly incident 

radiation (quantity and duration) for the month in which the test is being 

performed, with a cycle equivalent to the natural photoperiod.

(iii) Watering. 

(A) At least twice before test substance application (see paragraph 

(e)(3)(iv) of this guideline). Microcosms should be watered as dictated by 
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a predetermined water regime based on site history (see paragraph 

(e)(4)(i)(A)(3) of this guideline) with either reagent water or with 

rainwater that has been collected from the region of interest, filtered, and 

stored in a cooler at 4 degrees Celsius (

oC). The selected leaching regime 

(see paragraph (e)(3)(iv) of this guideline) should consider the watering 

regime. Care should be taken to provide sufficient water for normal plant 

functions without over watering.

(B) Water simulating rainfall or leaching should be filtered rainwater from 

the site being evaluated or reagent water with a known chemical 

composition.

(C) The volume of reagent water or rainwater required for laboratory 

microcosms may be determined on-site using a microcosm of the same 

soil type without vegetation. The volume selected should be identical for 

all microcosms and be sufficient to bring the A horizon of the soil surface 

to field capacity.

(9) Observations―

(i) Measurement of test substance. 

(A) Standard analytical methods, if available, should be used to establish 

actual concentrations of solutions of the test substance and should be 

validated before beginning the test. An analytical method is not 

acceptable if likely degradation products of the test substance, such as 

hydrolysis and oxidation products, cause positive or negative interference. 

The pH of these test solutions should also be measured before use.

(B) Samples of soil leachate, plant tissue (including roots and shoots), and 

soil from three depths should be analyzed for radioactivity, and 

identification and quantification of the test substance and its 

transformation products. The three soil depths should be selected based 

on soil sorption of the test substance and results from the range-finding 

test. These depths should be relatively close to the soil surface (1 to 2 cm) 

for radiolabeled chemicals that are strongly sorbed to soils. If any isotope 

appears in the leachate during the range-finding test, the depth selection 

should be lower in the soil profile. The entire soil layer should be taken as 

the sample, and then subsamples should be homogenized and extracted 

with solvents appropriate for the test substance. Additional extraction 

steps, such as acidification and extraction with non-polar solvents, Sohxlet 

extractions with polar and/or nonpolar solvents, alkaline or acid hydrolysis 

with or without heat, detergent extractions, or protease digestion may be 

necessary. The 14C in the soil or plant samples which cannot be extracted 

should be oxidized and analyzed as carbon dioxide (

14CO2) and reported 

as bound residue. Extracts and the oxidized or dissolved samples should 

be counted by 

14C liquid scintillation.
Page 12 of 22

(C) Identification and quantification of the test substance or its 

transformation products, expressed as a percent of the original application, 

in various compartments of the microcosm should be performed using gasliquid chromatography (GLC), thinlayer chromatography (TLC) or highpressure liquid chromatography (HPLC). TLC autoradiography using noscreen X-ray film for chromatographed fractions which are found to be 

radioactive by liquid scintillation counting may be most cost-effective. 

However, whenever possible, the identity of the test substance and its 

transformation products in fractions which are found to be radioactive by

liquid scintillation counting should be verified by GLC, HPLC, or other 

appropriate methods. Also, the concentration of the test substance and 

transformation products should be verified by an alternative 

chromatographic method (e.g., HPLC or GLC) with known standards.

(ii) Environmental conditions―

(A) Air temperature. Temperatures should be monitored continuously at 

the top of the plant canopy.

(B) Light intensity. Light intensity measurements should be taken daily, 

but should be taken at least at the beginning and end of the test. A

photosynthetically active radiation (PAR) sensor should be used to 

measure light quality. Additional information on the use of lighting in 

plant toxicity tests can be found in the references given in OCSPP

850.4000.

(C) Watering. Records should be kept noting the volume and days upon 

which a soil-microcosm is watered. Observations of possible moisture 

stress should be made and recorded daily.

(D) Pests. Daily observations should be made on pest pressure using an 

index of the extent of infestation. Pest infestation may affect the 

interpretation of study results and therefore should be adequately 

described. Frequency, methods, and rates used for treating an insect or 

disease should be recorded.

(iii) Measures of effect―

(A) Appearance and condition. Plants should be carefully monitored for 

changes in physical appearance, such as stunting, discoloration, or 

chlorosis and/or necrosis of the leaves.

(B) Plant primary productivity. To measure plant primary productivity, 

plants from the natural grassland or agricultural microcosm should be 

harvested at the end of the test period (a minimum of 12 weeks) and, 

possibly, once or twice during that period, depending on the type of plants 

grown and the extent of plant growth. For example, recommend sampling 

vigorously growing grasses during the middle of the test. Plants should be 
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clipped to approximately 2.5 cm above the soil surface. Harvested plants 

should be stored in separate paper bags for each microcosm, and air-dried, 

oven-dried, or both soon after harvest. The test may be extended beyond 

12 weeks to accommodate plant species that take longer to reach the 

desired maturity (e.g., seed production). Plant productivity, depending on 

the plant species, may be measured as total yield and/or yield by plant 

part, e.g., total biomass or grain. Minimally, plant productivity should be 

measured as air-dried and oven-dry weight expressed as grams per square 

meter; in the grassland microcosms, monocotyledons and dicotyledons 

should be separated for both plant productivity measurements and 

radiochemical assay.

(C) Nutrients in leachate. Nutrient losses should be sampled in soil 

leachates. Nutrients to be measured should be selected based on the 

properties of the test substance and the results of the range-finding test 

(see paragraph (d)(3)(iv) of this guideline) and should include calcium, 

potassium, nitrate-nitrogen, orthophosphate, ammonium-nitrogen, and 

dissolved organic carbon (DOC). Leachate should be collected in acidwashed, 500-mL flasks attached to the end of the Buchner funnel by inert 

plastic tubing. Leaching is induced by adding a volume of rainwater or 

reagent water above that necessary to bring the soil profile to field 

capacity. The volume of additional water needed to induce leaching in a 

specific core should be determined when the cores are extracted from the 

field. The volume of rainwater or reagent water needed should be 

recorded. Flasks to collect the leachate may be supported by a wooden 

board fastened under the microcosm cart. The volume of leachate should 

be recorded and the pH determined using a glass electrode. Samples 

should be centrifuged at low speed (e.g., 5,000 revolutions per minute 

(rpm)) and filtered through a 0.45 micrometer (μm) filter. The sample 

should be divided into two aliquots and stored in the dark at 4 oC with 

blanks consisting of reagent water and reference standards in quantities 

sufficient for instrument calibration. Standard techniques suitable for 

nutrient analysis may include atomic absorption spectrophotometry for 

calcium and potassium, and a Technicon Autoanalyzer II for nitrate 

nitrogen, orthophosphate, dissolved organic carbon (DOC), and 

ammonium nitrogen.

(D) Soil invertebrate and microbe diversity and abundance. Soil 

invertebrates and microbes may be sampled at the end of the test.

(f) Treatment of results―

(1) Experimental design. Analysis of variance (ANOVA) calculations should be 

performed to test for position effects within the carts and within the environmental area 

where the test is performed. If these tests are significant at the 5% level (P < 0.05), this 

should be accounted for in subsequent statistical analyses.
Page 14 of 22

(2) Plant productivity. 

(i) The effects of different concentrations of the test substance on productivity can 

be evaluated initially by using side-by-side histograms displaying calculated 

means (expressed as grams per square meter), variances, 95% confidence 

intervals, and two standard errors for air- and oven-dried biomass collected from 

control and treatment groups. Early evaluation will indicate whether logarithmic 

or some other transformation of the data is necessary for graphic display and 

analysis. Pair-wise comparisons may be necessary for variables which were 

measured only once during the 12-week test.

(ii) Biomass data should be analyzed by ANOVA and least significant differences 

multiple-range procedures. The level of significance for all tests should be at the 

5% level. Where treatment effects and interactions between and among various 

factors are important, a two-way ANOVA or factorial analysis should be 

performed.

(iii) Regression/correlation analysis should be performed on plant productivity 

results. Obvious recording or reporting errors in the data should be excluded but 

noted in the final report. If substantial data are excluded, deficiencies in quality 

control may necessitate repeating the test. Once outlying values have been 

detected and removed from further statistical evaluations, regression models or 

probit analysis should be used to estimate the concentration at which 50% of the 

productivity observed in controls occurred in the treated groups (IC50). Ordinary 

linear least-squares regression analysis should initially be performed, and 

predicted responses in each group should be compared using a Student t-test (onesided). If productivity appears to be bimodal when compared to controls, a twosided Student t-test may be necessary. It may also be necessary to transform the 

data or fit a quadratic or cubic least-squares-regression model to the data for this 

type of response. Positional effects should be included in the data. Computer 

software packages may be useful.

(3) Plant injury. Statistical analyses of the effects of the test substance and 

transformation products on the appearance of plants are not necessary unless there is a 

clearly identifiable pattern of effects. If deemed necessary, types of injury should be 

ranked by severity. A non-parametric test, such as the Kruskal-Wallis test, should then 

be performed.

(4) Nutrient losses. 

(i) Based on the nutrients selected for analysis in soil leachate, the total 

cumulative loss of each nutrient from each microcosm should be calculated by

multiplying the concentration of the nutrient collected at each sampling time by 

the total volume leached from that microcosm for that collection date and adding 

the product to the previous sum of total loss.
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(ii) Means (± standard error) of the cumulative nutrient losses for each treatment 

concentration for each collection date should be plotted as a function of days after 

seeding for the agricultural microcosm or days after application of the test 

substance for the natural grassland microcosm. Zero loss should be the starting 

point. If there was no leachate for any microcosm during a particular collection 

period, the data point should be recorded as zero so that no data are considered 

missing.

(iii) A one-way ANOVA should be performed on total cumulative nutrient loss 

data at the end of the test, to evaluate effects of different concentrations of the test 

substance. A multiple-range procedure, such as Duncan's, should be used to 

determine which specific treatment means are different from each other.

(iv) Regression and/or correlation analysis comparing losses of each nutrient 

analyzed versus plant productivity should be performed.

(5) Chemical fate analysis. 

(i) At the end of the test, the mass balance or final distribution of the test 

substance and its transformation products in above- and below-ground plant 

tissues, selected depths through the soil profile, and losses through soil leaching 

and gaseous transport should be calculated for each concentration of the substance

tested.

(ii) Calculations should be based on measured radioactivity in a specific 

compartment of the microcosm, on a per-gram basis, times the total weight or 

volume of test substance in that compartment, expressed as dry weight when 

appropriate. All calculations should be corrected for radioactive decay (as 

appropriate) that has occurred since the beginning of the test. Quantities of the 

test substance and its transformation products should be expressed as a percent of 

the original application of the test substance.

(iii) Statistical analyses should be performed for each exposure concentration on 

any differences in distribution of the test substance in the primary compartments 

of the microcosm and in soil leachate. Multicompartmental modeling and 

multivariate analysis of variance may also prove useful in assessing the fate of a 

test substance and its transformation products.

(iv) The time to reach steady-state loss through leaching and the time to initiate 

leaching should be calculated for each exposure concentration.

(6) Radioactivity budget. Calculation of a complete mass balance of all radioactivity 

should be performed as in paragraphs (f)(6)(i) of this guideline:

(i) Total radioactivity added per microcosm should be calculated based on the 

decay rate of the radioactive label (e.g.,

14C), the total amount of radioactive label 

added to the test substance initially, the length of time between formulation and 
Page 16 of 22

microcosm exposure (radioactive decay), and the particular concentration of the 

test substance added to the microcosm.

(ii) Total radioactivity removed from the microcosm should be calculated based 

on the data in paragraphs (f)(6)(ii)(A) through (f)(6)(ii)(C) of this guideline:

(A) Soil leachate concentration times the volume of soil leachate lost per 

collection date.

(B) Calculated gaseous losses of the test substance.

(C) The type of radiolabel and rate of radioactive decay of that label 

during the test.

(iii) Total radioactivity remaining in the microcosm can be calculated based on 

analysis of the radioactivity in each of the primary compartments in paragraphs 

(f)(6)(iii)(A) through (f)(6)(iii)(C) of this guideline:

(A) Above-ground plant tissues.

(B) Below-ground plant tissues, i.e., cleaned of soil particles.

(C) The soil profile.

(7) Bioconcentration. The ratio of the amount of radioactivity in above-ground plant 

tissues to the amount in the top 15 cm of soil should be calculated on a concentration-perunit, dry-weight basis. Side-by-side histograms of the BCFs should be compared for 

statistical differences.

(8) Abundance and distribution of soil organisms. Appropriate statistical methods 

should be used to evaluate the distribution and abundance of soil invertebrates and 

function of the soil microbial community with respect to treatment concentrations.

(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.
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Table 1.—Summary of Test Conditions for a Soil-Core Microcosm Test

Test duration At least 12 weeks (extend to accommodate plant 

species which take longer to reach desired 

maturity (e.g., seed production))

Temperature Approximates outdoor temperature during the 

growing season in region of interest

Light quality, intensity and photoperiod Simulates outdoor light conditions during the 

growing season in region of interest

Watering Watered as dictated by a predetermined water 

regime based on site history using site rainwater 

or reagent-water. (For soil leachate collection, at 

least twice before test substance application, then 

once every 2 or 3 weeks.)

Test chamber size 60 cm long polyethylene (e.g., Driscopipe) tube, 

17 cm diameter

Number of replicate chambers per test treatment Ten

Number of test concentrations At least three, plus appropriate controls

Test organisms Agricultural microcosm: A mixture of grasses and 

broad-leaved plants that is typically grown 

together as an agricultural crop in the region of 

interest.

Natural grassland microcosm: Vegetation covering 

the natural grassland ecosystem of interest.

Administration of test substance In water, directly to soil on top of microcosm, or 

sprayed onto plants, as appropriate to simulate 

actual use or exposure

Measures of effect (measurement endpoints) Productivity of plants (e.g., biomass) NOEC (and 

LOEC), plant injury NOEC (and LOEC), nutrient 

losses NOEC (and LOEC), regression analysis of 

plant biomass against nutrient losses, fate of test 

substance, BCF, and effects on soil organisms 

(optional)

(h) Test validity elements. 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 850.4000, it is unlikely a study will be rejected when there are 

slight variations from guideline environmental conditions and study design unless the controls 

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 any 

resulting effects on test endpoints noted and discussed. 
Page 18 of 22

Table 2. — Test Validity Elements for Soil-Core Microcosm Test

1. The total amount of test substance applied to each replicate within a treatment concentration was not 

the same.

2. Controls (and vehicle controls, if applicable) were not included in the test.

3. Less than three concentrations of the test substance were used.

(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 850.4000.

(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)), radiolabeling if any, location of label(s), 

and radiopurity.

(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 and the treatment doses used in 

the range-finding and definitive test.

(iv) If a vehicle (e.g., solvent, dust) is used to prepare stock or test substance

provide: 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 dose(s) 

used in the treatments.

(v) Stability of the test substance and, if used, control substances under the 

conditions of administration.

(4) Soil-core microcosm. 

(i) Type, source, and date of soil-core extraction.

(ii) Method and equipment used to extract the soil core.

(iii) A description of soil-core dimensions: diameter, depth, depth of top soil 

removed and later backfilled (if agricultural soil-core).
Page 19 of 22

(iv) Chemical and physical characteristics: texture or type of soil, soil components 

and profile, soil particle distribution, percent organic matter, percent organic 

carbon, pH, soil density, and mass of soil.

(v) Biota―

(A) Natural grassland soil-core. 

(1) Identification of plants included in the natural vegetation: 

scientific and common name, plant family.

(2) For each soil-core, the density of plants by plant species at test 

initiation.

(3) Height, development stage, root depth and condition of plants 

in each soil-core at test initiation.

(B) Agricultural soil-core. 

(1) Identification of plants: scientific and common name, plant 

family, and variety.

(2) Test date of germination rating and germination percentage.

(3) History of the seed: source, name of supplier, seed year or 

growing season collected, batch or lot number, seed treatment(s), 

and storage conditions.

(4) Seed size class.

(5) Planting procedures and any special handling of seed before 

planting.

(6) Number of total weight (for smaller species) of seeds tested per 

concentration (in agricultural microcosm).

(5) Test system and conditions. Description of the test system and conditions used in 

the definitive test, and any preliminary range-finding tests.

(i) A description of the test system, including type of greenhouse or 

environmental chamber conditions: type, size, location.

(ii) Description of soil core support system and the leachate collection system: 

type, material, dimensions.

(iii) Number of microcosms per treatment level.

(iv) Randomization procedures used to position microcosms and assign test 

concentrations to particular microcosms.
Page 20 of 22

(v) Methods for preparing the test treatments: application methods (including 

equipment type and method for calibrating the application equipment), 

information about any solvent used to dissolve and apply the test substance, the 

test substance concentrations or doses (total mass and radioactivity) applied to an 

individual soil-core microcosm.

(vi) Description of the water used for the test: source, special treatments, and 

chemical composition such as hardness, pH, nutrients, suspended solids, total 

organic carbon, unionized ammonia, residual chlorine, total organophosphate 

pesticides, total organochlorine pesticides, and total organic chlorine.

(vii) Description of the watering schedule for the microcosms, the method of 

watering, and the volume and rate of water applied for each microcosm.

(viii) Number of test substance applications and dates applied for a microcosm.

(ix) The photoperiod and light source.

(x) Methods and frequency of environmental monitoring performed during the 

definitive study for air temperature, humidity, and light intensity.

(xi) Leachate collection methods, frequency, dates of collection, amount collected 

by microcosm.

(xii) Number of soil samples collected, depth interval in the soil profile collected 

for each sample, and moisture content of each sample by microcosm and 

treatment for analysis of test substance and transformation products.

(xiii) Frequency, duration, and methods of observations on microcosm biota.

(xiv) Frequency of collection of vegetative matter by microcosm and treatment 

for analysis of test substance and transformation products and biomass (dry

weight).

(xv) For the definitive test, all analytical procedures 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 (air temperature, humidity and light 

intensity) in tabular form (provide raw data for measurements not made on a 

continuous basis), and descriptive statistics (mean, standard deviation, minimum, 

maximum).

(ii) Results of the range-finding test and measurements.

(iii) Results of the definitive test including:
Page 21 of 22

(A) Tabular summary by microcosm and treatment of visible effects of the 

test substance on intact plants (include raw data). A description of the 

phytotoxicity rating system used should be included.

(B) Total productivity and/or yield by plant part (e.g., total biomass or 

grain) expressed as grams per square meter oven-dry weight by

microcosm and treatment (include raw data) and the mean and standard 

deviation of biomass by plant part for each treatment.

(C) Side-by-side histograms displaying calculated means (expressed as 

grams per square meter) for air- and oven-dried biomass.

(D) Tabular summary by microcosm, treatment, and date of losses of 

selected nutrients in soil leachate expressed as grams per liter of leachate, 

and the volume of leachate collected (include raw data) and the mean and 

standard deviation of the losses by treatment.

(E) Means (± standard error) of the cumulative nutrient losses for each 

treatment concentration for each collection date should be plotted as a 

function of days after seeding for the agricultural microcosm or days after 

application of the test substance for the natural grassland microcosm. 

Zero loss should be the starting point.

(F) Tabular summary by microcosm, treatment, and soil depth of test 

substance and transformation products in soil samples expressed in grams 

per dry weight (include raw data) and the mean and standard deviation by

treatment and soil depth.

(G) Tabular summary of the radioactivity budget including total 

radioactivity added to, removed from (via soil leaching, gaseous transport, 

and radioactive decay), and remaining in each microcosm (plant tops and 

roots and selected soil depths) (include raw data). Losses via gaseous 

transport should be estimated and expressed as milligrams per cubic 

meter.

(H) Tabular summary of the percent distribution of the test substance and 

its transformation products in the primary compartments of the 

microcosm, including above- and below-ground plant tissues and selected 

depths through the soil profile.

(I) Bioconcentration of the test substance in above-ground plant tissue 

expressed as the ratio of the concentration in plant tissue to the 

concentration in the top 15 cm of dry soil for each microcosm by

treatment (include raw data).

(J) Side-by-side histograms of the treatment mean calculated BCFs by

plant part.
Page 22 of 22

(iv) Results of the analysis of data for each measure of effect (total productivity, 

nutrient loss, soil profile concentrations of test substance and transformation 

products, distribution of test substance and transformation products in primary 

compartments of the microcosm, and plant tissue BCFs) should include the results 

of ANOVA and multiple range tests.

(v) Results of regression analysis (concentration-response curve fitting) for total 

productivity, nutrient loss, the IC50 values, slope, intercept, 95% confidence 

limits, the results of a goodness-of-fit test, e.g., chi-square test.

(vi) A description of all statistical methods, including: software used, handling of 

outlier data points, handling of non–detect or zero values, tests to validate the 

assumptions of the analyses, level of significance, any data transformations or 

operations performed on the data, for hypothesis tests a measure of the sensitivity

of the test (either the minimum significant difference or the percent change from 

the control that this minimum difference represents. 

(j) References. The references in this paragraph should be consulted for additional background 

material on this test guideline.

(1) Hammons, A.S. 1981. Methods for Ecological Toxicology: A Critical Review of 

Laboratory Multispecies Tests. EPA-560/11-80-026

(2) Hammons, A.S. 1981. Ecotoxicological Test Systems: Proceedings of a Series of 

Workshops. EPA-560/6-81-004. 

(3) Van Voris, P., Tolle, D.A., and Arthur, M.F. 1985. Experimental Terrestrial SoilCore Microcosm Test Protocol. A Method for Measuring the Potential Ecological 

Effects, Fate, and Transport of Chemicals in Terrestrial Ecosystems. EPA/600/3-85/047, 

PNL-5450, Corvallis Environmental Research Laboratory, Corvallis, OR.