
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
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  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.
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  	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.
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  	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 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, 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 (ug/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/cm[3]) 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 ug/g which totals 1 ug/g or a single dose of 1 ug/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 ug/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 site-specific 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 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 ([14]C), nitrogen-15 ([15]N), 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(R) (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.
                  
                  (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.
               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 range-finding 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 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 pH-adjusted, 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.
                        (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 range-finding 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 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(R) 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.  
            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 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 ([o]C).  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 [14]C in the soil or plant samples which cannot be extracted should be oxidized and analyzed as carbon dioxide ([14]CO2) and reported as bound residue.  Extracts and the oxidized or dissolved samples should be counted by [14]C liquid scintillation.
                  (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 gas-liquid chromatography (GLC), thinlayer chromatography (TLC) or high-pressure liquid chromatography (HPLC).  TLC autoradiography using no-screen 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 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 acid-washed, 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 [o]C 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.
      (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 (one-sided).  If productivity appears to be bimodal when compared to controls, a two-sided 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.
            (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.,[14]C), the total amount of radioactive label added to the test substance initially, the length of time between formulation and 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-per-unit, 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.

     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. 
       	Table 2.  --  Test Validity Elements for Soil-Core Microcosm Test
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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.
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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).
            (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.
            (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:
                  (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.
            (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 Soil-Core 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. 
