United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-143
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.2450
Terrestrial (Soil-Core)
Microcosm Test
I

Public Draft"

-------
Introduction
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
i

-------
OPPTS 850.2450 Terrestrial (soil-core) microcosm test.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is the OPPT guideline under 40 CFR
797.3775 Soil-Core Microcosm Test (proposed in the Federal Register
of September 28, 1957 (52 FR 36363)).
(b)	Purpose. This guideline is intended for use in developing data
on the toxicity and fate of chemical substances and mixtures ("test sub-
stances") subject to environmental effects test regulations under the Toxic
Substances Control Act (TSCA) (Pub. L. 94-406, 96 Stat. 2083, 14 U.S.C.
2801 et seq.). This guideline prescribes tests using soil-core microcosms
to provide information on the potential fate and ecological effects of chem-
ical substances released to a specific terrestrial ecosystem. The United
States Environmental Protection Agency (EPA) will use data from these
tests in assessing the hazard of a test substance to the environment.
(c)	Definitions. The definitions in section 3 of TSCA and Part 792—
Good Laboratory Practice Standards of this chapter, apply to this test
guideline. The following definitions also apply:
Bioconcentration factor (BCF) means the ratio of the concentration
of test substance in plant tissue (i.e., biota) to that in soil.
Biota means 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.
Carrier means the organic solvent, solubilizer and/or other substance
used to disperse the test substance into microcosm water.
Soil-core means an intact, undisturbed (nonhomogenized) core 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 crops selected, without causing the plants to become sig-
nificantly root bound.
Soil-core microcosm means a physical miniaturized model of an inter-
acting community of autotrophs, omnivores, herbivores, carnivores, and
decomposers within an intact soil profile.
(d)	Test procedures—(1) Summary of the test. The purpose of the
soil-core microcosm test is to determine the potential fate and ecological
effects of a chemical substance, including its transformation products, re-
leased to a specific terrestrial ecosystem. A soil core, as shown in the
1

-------
following Figure 1, containing biota typical of the region of interest, is
treated with the test substance under controlled conditions in either a
growth chamber or greenhouse. The test is usually continued for a mini-
mum of 12 weeks from first application of the test substance to final har-
vest. 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 sub-
stances 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.
Figure 1.—Microcosm Structure and Materials
(2) Application of the test substance, (i) Whenever possible, the
test substance should be radiolabeled. The label may be 14C, stable iso-
2

-------
topes such as 15N, or other suitable labels and, if possible, should be lo-
cated 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, each component must be labeled and studied separately.
(ii) The test substance should be applied in a form which is reason-
ably consistent with the form in which it is expected to be released into
the environment. The method and pattern of application should also reflect
the actual or predicted field situation.
(A)	If the primary mode of exposure to the test substance is antici-
pated to be by addition of pH-adjusted, reverse osmosis (RO) water or
rainwater containing appropriate concentrations of the substance, the fol-
lowing procedure is recommended.
(7) Test substances which are likely to be released into the environ-
ment 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 purified (i.e., reverse osmosis) untreated
laboratory water with a known chemical composition.
(3)	The volume of reverse osmosis (RO) 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.
(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 carrier
is necessary, acetone or ethanol should be considered; however, the use
of carriers 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:
(7) If the test substance is likely to be a contaminant of irrigation
water, it should be applied periodically, such as daily or weekly, in propor-
tionate concentrations, such that the total amount applied equals the de-
sired level of treatment.
(2) 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.
3

-------
(3) 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 should be terminated at least 8 weeks after plants are sprayed.
(C) Water solubility and dissociation constant(s) of the test substance
and soil pH must be considered in determining the formulation of the test
substance. The treatment concentrations should bracket the known or ex-
pected 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.
(3) Range-finding test, (i) A range-finding test may be conducted
if definitive testing is necessary and to determine the concentrations of
test substance to be used in the definitive test.
(ii)	Physicochemical information supplied for the test substance
should be used to tailor the general range-finding test procedures to the
specific substance.
(iii)	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 change in plant growth or a 50 percent change in bacterial growth/
respiration should be tested. Also, the lowest concentration to be used
should not be lower than a factor of 10 times the analytical detection limit
in soil and 100 times the analytical detection limit in water.
(iv)	The range-finding test should last at least 4 weeks from first
application of the test substance to plant harvest. At the beginning of the
test, microcosms should be treated with a minimum of five concentrations
of the test substance. Three replicate microcosms are used for each con-
centration and for the control, resulting in a total of at least 18 microcosms.
Concentrations typically used for treatment are 0.1, 1.0, 10, and 100 mg/
L (ppm), if actual environmental concentrations are not known and cannot
be predicted. If appropriate, 1,000 |ig of test substance per gram of top
15 cm of dry soil should also be used. The bulk density (g/cm3) of the
dry soil should be used to calculate the exposures. Depending on the ex-
pected mode of release of the test substance, each recommended con-
centration may be applied as a single dose or may be divided into multiple
doses. In either case, the total amount of test substance applied for any
given concentration must be the same.
4

-------
(v) Each microcosm cart, in the following Figure 2, holding one rep-
licate of each test substance concentration and a control, should be moved
once per 7 days in the greenhouse to minimize location-induced effects.
Figure 2.—Arrangement of Microcosms in Styrofoam Cart
Soil-Core
(vi)	Losses of calcium, potassium, nitrate-nitrogen, orthophosphate,
ammonium-nitrogen, and dissolved organic carbon (DOC) should be meas-
ured in soil leachates. 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 RO 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 RO water needed should be recorded. Flasks to collect the
leachate may be supported by a wooden board fastened under the micro-
cosm cart. The volume of leachate should be recorded and the pH deter-
mined using a glass electrode. Samples should be centrifuged at low speed
(e.g., 5,000 rpm) and filtered through a 0.45-(im filter. The sample should
be divided into two aliquots and stored in the dark at 4 °C with blanks
consisting of distilled water and reference standards in quantities sufficient
for instrument calibration.
(vii)	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
5

-------
samples at the end of the definitive test should be taken nearer the top
of the soil column.
(viii) If no discernible effects of the test substance are detected during
the range-finding test at one-half of saturation or 1,000 (ig/g (whichever
is higher), including visible effects of plant injury, no definitive test is
necessary.
(4) Definitive test, (i) The purpose of the definitive test is to deter-
mine the potential fate and ecological effects of a test substance, including
its transformation products, in a site-specific natural grassland or agricul-
tural ecosystem.
(ii)	Chemical substances with high vapor pressures or high Henry's
law constants should not be tested in the soil-core microcosm as prescribed
in this guideline.
(iii)	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 spe-
cially designed, steel extraction tube, as shown in the following Figure
3, and a backhoe. Disturbances during extraction and preparation of the
soil core should be minimized.
6

-------
Figure 3.—Diagram of Microcosm Extraction Tube
(iv)(A) For an agricultural microcosm, the soil which is plowed (gen-
erally, 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 as demonstrated in Figure 1
in paragraph (d)(1) of this guideline. The Driscopipe should then contain
a 45-cm core of subsoil. 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) A mixture of grasses and broad leaves (e.g., legumes) should be
included in the agricultural microcosm. 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. The rate of
7

-------
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.
(v)(A)	For a natural grassland microcosm, the vegetation covering
the natural grassland ecosystem should be clipped to a uniform height be-
fore the core is extracted. Natural plant cover should be sufficiently diverse
to be representative of plant species in the region of interest.
(B) The soil core from the grassland ecosystem should be removed
as a single unit (soil and Driscopipe) from the extraction tube, taken to
the laboratory, and placed on a Buchner funnel covered by a thin layer
of glass wool. The funnel and tube should be washed with acid (50 percent
concentrated HNO3) before use and then rinsed with RO water.
(vi)	Six microcosms are typically contained in a moveable cart which
is packed with styrofoam beads, as shown in Figure 2 in paragraph
(d)(3)(v) of this guideline.
(vii)	An appropriate random process should be used, such as com-
pletely randomized, randomized block, or Latin-square design, to assign
microcosms to different concentrations of the test substance.
(viii)	The method and pattern of application and the form of the test
substance used should approximate a reasonable scenario of how the sub-
stance is expected to be released at the site in question (see paragraph
(d)(2)(ii) of this guideline).
(ix)	At the beginning of the test, microcosms should be treated with
at least three concentrations of the test substance. Ten replicate micro-
cosms should be used for each of the three concentrations and for the
control, for a total of 40 microcosms. The treatment concentrations should
be selected to produce a 20 to 25 percent change in plant productivity
in each treatment based on results from the range-finding test. The 10
replicate microcosms in each treatment group should be used as five rep-
licate pairs.
(x)	Microcosms that have been paired for analysis should be placed
in different carts to ensure that environmental conditions are as uniform
as possible.
(xi)	The structure, materials, and treatment of control microcosms
should be the same as that of exposed microcosms except that none of
the test substance is applied.
(xii)	Microcosms should be watered as dictated by a predetermined
water regime based on site history with either reverse osmosis (RO) water
or with rainwater that has been collected from the region of interest, fil-
tered, and stored in a cooler at 4 °C. Care should be taken to provide
sufficient water for normal plant functions without overwatering. If the
8

-------
test substance is applied as an aerosol or powder, plants should be sprin-
kled immediately after treatment to avoid resuspension of particulates and
reduce the potential for cross-contamination of exposure concentrations.
(xiii)	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 col-
lection and analysis. Microcosms should be leached, as described in para-
graph (d)(3)(vi) 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 percent more soil cores should be extracted than
are required for the tests. When the microcosms are leached before plant-
ing, 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.
(xiv)	Light intensity measurements should be taken daily, but should
be taken at least at the beginning and end of the test.
(xv)	Temperatures should be monitored continuously at the top of
the plant canopy.
(xvi)	Plants should be carefully monitored for changes in physical
appearance, such as stunting, discoloration, or chlorosis and/or necrosis
of the leaves.
(xvii)	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 types of plants growth. For example, vigor-
ously growing grasses may be sampled during the middle of the test. Plants
should be clipped to approximately 2.5 cm above the soil surface. Har-
vested 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 ex-
tended beyond 12 weeks to accommodate plant species which 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 productiv-
ity should be measured as 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.
(xviii)	Nutrient losses should be sampled in soil leachates. Nutrients
to be measured should be selected based on the properties of the test sub-
9

-------
stance and the results of the rangefinding test (see paragraph (d)(3)(vi)
of this guideline).
(xix)	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. 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 sub-
stance. 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 extrac-
tions, or protease digestion may be necessary. The 14C in the soil or plant
samples which cannot be extracted should be oxidized and analyzed as
14C02 and reported as bound residue. Extracts and the oxidized or dis-
solved samples should be counted by 14C liquid scintillation.
(xx)	Soil invertebrates and microbes may be sampled at the end of
the test.
(5) Analytical measurements—(i) Chemical. (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 prod-
ucts 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)	The fate or final distribution of the test substance and its trans-
formation products should be determined by methods appropriate to the
test, including sensitivity factors adequate to verify exposure and distin-
guish between the test substance, its transformation products, and naturally
occurring materials present in the test system. Whenever possible, this
should involve use of a radiolabeled test substance, and subsequent analy-
sis of the primary microcosm compartments and soil leachate for radio-
activity and chemical identity.
(C)	Identification and quantification of the test substance or its trans-
formation 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.
10

-------
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.
(D) Standard techniques suitable for nutrient analysis may include
atomic absorption spectrophotometry for calcium and potassium, and a
Technicon Autoanalyzer II for nitrate nitrogen, orthophosphate, DOC, and
ammonium nitrogen.
(ii) Numerical—(A) 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 per-
formed. If these tests are significant at the 5-percent level (P < 0.05),
this should be accounted for in subsequent statistical analyses.
(B) Productivity. (7) 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-percent confidence intervals, and two stand-
ard errors for air- and oven-dried biomass collected from control and treat-
ment groups. Early evaluation will indicate whether logarithmic or some
other transformation of the data is necessary for graphic display and analy-
sis. Pair-wise comparisons may be necessary for variables which were
measured only once during the 12-week test.
(2)	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-percent level. Where treatment effects and inter-
actions between and among various factors are important, a two-way
ANOVA or factorial analysis should be performed.
(3)	Regression/correlation analysis should be performed on plant pro-
ductivity 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 out-
lying values have been detected and removed from further statistical eval-
uations, regression models or probit analysis should be used to estimate
the concentration at which 50 percent of the productivity observed in con-
trols occurred in the treated groups (EC50). 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 trans-
form 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
11

-------
in the data. Computer software packages such as SAS (Statistical Analysis
System) or BMCP (Biomedical Computer Program) may be useful.
(C)	Plant injury. Statistical analyses of the effects of the test sub-
stance 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.
(D)	Nutrient losses. (7) Based on the nutrients selected for analysis
in soil leachate, the total cumulative loss of each nutrient from each micro-
cosm 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.
(2)	Means (+SE) 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.
(3)	A one-way ANOVA should be performed on total cumulative nu-
trient loss data at the end of the test, to evaluate effects of different con-
centrations of the test substance. A multiple-range procedure, such as Dun-
can's, should be used to determine which specific treatment means are
different from each other.
(4)	Regression and/or correlation analysis comparing losses of each
nutrient analyzed versus plant productivity should be performed.
(E)	Chemical fate analysis—(7) 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.
(2)	Calculations should be based on measured radioactivity in a spe-
cific 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 radio-
active 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.
(3)	Statistical analyses should be performed for each exposure con-
centration on any differences in distribution of the test substance in the
12

-------
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 trans-
formation products.
(4) The time to reach steady-state loss through leaching and the time
to initiate leaching should be calculated for each exposure concentration.
(F)	Radioactivity budget. Calculation of a complete mass balance
of all radioactivity should be performed as follows:
(7) 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 microcosm exposure (radioactive decay), and the
particular concentration of the test substance added to the microcosm.
(2)	Total radioactivity removed from the microcosm should be cal-
culated based on the following data:
(z) Soil leachate concentration times the volume of soil leachate lost
per collection date.
(zz) Calculated gaseous losses of the test substance.
(z/z) The type of radiolabel and rate of radioactive decay of that label
during the test.
(3)	Total radioactivity remaining in the microcosm can be calculated
based on analysis of the radioactivity in each of the following primary
compartments:
(z) Above-ground plant tissues.
(zz) Below-ground plant tissues, i.e., cleaned of soil particles.
(z/z) The soil profile.
(G)	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.
(H)	Soil organisms. Appropriate statistical methods should be used
to evaluate the distribution and abundance of soil invertebrates and func-
tion of the soil microbial community with respect to treatment concentra-
tions.
(e) Test conditions—(1) Test species—(i) Selection. Biota should
be included in the microcosm. A mixture of two or three species of grasses
or broad leaves, such as legumes, representative of the area or region
13

-------
where the test substance is expected to be released or applied to crops
or soil, should be included in the agricultural microcosm. 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.
(ii) Seed selection. 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.
(2) Facilities—(i) Apparatus. (A) The greenhouse or growth cham-
ber should provide adequate environmental controls to meet light and tem-
perature specifications.
(B)	Laboratory facilities for test substance determinations should in-
clude: 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 deter-
minations, drying ovens, refrigerators, and standard laboratory glassware.
(C)	A specially designed steel extraction tube and a backhoe are need-
ed to extract soil cores.
(ii)	Containers and supporting equipment. (A) For the definitive
test, at least 18 microcosms are required. The three basic materials used
for a single microcosm are: A 60-cm long Driscopipe tube (17.5 cm diam-
eter), a 186 mm-diameter porcelain Buchner funnel, and a thin layer of
glass wool (see Figure 1 in paragraph (d)(1) of this guideline). Containers
used in each test should be of equal size and volume and possess the
same configuration.
(B) Three mobile carts should be used to hold 18 microcosms. The
carts should be designed to hold adequate styrofoam beads for insulation
in Figure 2 under paragraph (d)(3)(v) of this guideline.
(iii)	Cleaning. All equipment used in the test should be cleaned be-
fore use and should be washed according to good standard laboratory prac-
tices, to remove any residues remaining from manufacture or use. A di-
chromate solution should not be used for cleaning containers. Disposal
of all detergents and acids that have been used to clean the Driscopipe
funnels, and laboratory glassware, and disposal of all liquid and solid sam-
14

-------
pies and remaining undisturbed portions of the test system should conform
to applicable existing Federal regulations.
(3) Test parameters. Microcosms should be kept in a greenhouse
or environmental chamber with controlled environmental conditions.
(i)	The temperature should approximate outdoor temperatures that
occur during a typical growing season in the region of interest.
(ii)	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 envi-
ronmental 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.
(f) Reporting. The report should include, but not necessarily be lim-
ited to, the following information:
(1)	Name and address of the facility performing the study and the
dates on which the study was initiated and was completed, terminated,
or discontinued.
(2)	Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.
(3)	Statistical methods used for analyzing the data.
(4)	The test substance identified by name, Chemical Abstracts Service
(CAS) registry number or code number, source, lot or batch number,
strength, purity, and composition or other appropriate characteristics, such
as water solubility and vapor pressure at 25 °C.
(5)	Stability of the test substance and, if used, control substances
under the conditions of administration.
(6)	A description of the methods used, including:
(i)	Greenhouse or environmental chamber conditions, including type
and size, temperature, photoperiod, and light intensity.
(ii)	Source, any special treatment, and chemical composition of the
water used.
(iii)	Method and equipment used to extract the soil core.
(iv)	Randomization procedures used to position microcosms and as-
sign test concentrations to particular microcosms.
15

-------
(v) Frequency, duration, and methods of observations.
(7)	A description of the test system used including:
(i)	The soil core, including chemical, biological, and physical charac-
teristics, source, soil type, and when applicable, identification of plant spe-
cies included in the natural vegetation or the scientific names and sources
of the agricultural plants selected and histories of the species, e.g., percent-
age of seeds germinating and seed size class.
(ii)	Planting procedures and any special handling of seed before plant-
ing.
(iii)	Number of total weight (for smaller species) of seeds tested per
concentration (in agricultural microcosm).
(8)	A description of the experimental design, test substance concentra-
tions, method and pattern of application, replicates, controls, and carriers.
The reported results should include:
(i)	Results of the range-finding test and measurements.
(ii)	Results of the definitive test including:
(A)	Visible effects of the test substance on intact plants.
(B)	Total productivity and/or yield by plant part (e.g., total biomass
or grain) expressed as grams per square meter oven-dry weight.
(C)	Losses of selected nutrients in leachates.
(D)	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
expressed as dry weight, and in soil leachate expressed as grams per square
meter. Losses via gaseous transport should be estimated and expressed as
milligrams per cubic meter.
(E)	A radioactivity budget including total radioactivity added to, re-
moved from (via soil leaching, gaseous transport, and radioactive decay),
and remaining in each microcosm (plant tops and roots and selected soil
depths).
(F)	Bioconcentration of the test substance in above-ground plant tis-
sue expressed as the ratio of the concentration in plant tissue to the con-
centration in the top 15 cm of dry soil.
(9)	A description of all circumstances that may have affected the qual-
ity or integrity of the data.
16

-------
(10)	The name of the sponsor, study director, principal investigator,
names of other scientists or professionals, and the names of all supervisory
personnel involved in the study.
(11)	A description of the transformations, calculations, or operations
performed on the data, a summary and analysis of the data, and a statement
of the conclusions drawn from the analysis. Results of the analysis of data
should include the concentration response curves with 95-percent con-
fidence limits, the results of a goodness-of-fit test, e.g., X2 test, and EC50s.
(12)	The signed and dated reports of each of the individual scientists
or other professionial involved in the study including each person who,
at the request or direction of the testing facility or sponsor, conducted
an analysis or evaluation of data or specimens from the study after data
generation was completed.
(13)	The location where all specimens, raw data, and the final report
are stored.
(14)	The statement prepared and signed by the quality assurance unit.
17

-------