United States      Prevention, Pesticides     EPA712-C-96-161
          Environmental Protection    and Toxic Substances     April 1996
          Agency        (7101)
&EPA   Ecological Effects Test
          OPPTS 850.5100
          Soil Microbial
          Community Toxicity Test
                'Public Draft"

     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

     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
(7U.S.C. I36,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:

     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,  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

OPPTS 850.5100  Soil microbial community toxicity 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 40 CFR 797.3700 Soil Microbial Com-
munity  Toxicity Test (proposed  in the FEDERAL REGISTER of September
28, 1987 (52 FR 36350)).

     (b) Purpose.  This guideline is  intended for use  in developing data
on the toxicity of chemical substances and  mixtures ("test substances")
subject  to environmental effects  test regulations.  The guideline prescribes
a test using natural soil samples to develop data  on  the toxicity of test
substances to microbial populations indigenous to the  soil.  The 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 40 CFR
Part 792—Good Laboratory Practice Standards apply to this  guideline. The
following definitions also apply:

     Ammonification is conversion of organic nitrogen compounds to am-
monia (NH3) or to ammonium ion (NH4) compounds, performed by a vari-
ety of microorganisms in soil and water.

     Carbon dioxide (CO2) efflux is the evolution of  CCh  gas from sub-
strates mineralized by microbial action—indicative of respiration.

     EC X is the experimentally-derived test substance concentration that
is calculated to affect X percent of the test species.

     MoPascal (kPa) is a unit of pressure in the  meter-kilogram-second
system  equivalent to one newton per square meter (i.e., 1 Pa x  1,000)
used as  a measure of water availability in soils.

     Mineralization is the  complete or ultimate degradation  by microorga-
nisms of organic material  to form inorganic  end-products, e.g., carbon di-
oxide, water, chloride, ammonium, nitrates, or orthophosphate.

     Nitrification is the oxidation of ammonium salts to nitrites (NCh) and
nitrates  (NOs), performed by relatively specialized microorganisms.

     Surface soil is that layer of soil representing the top  15 cm of the
area to be sampled, excluding  the litter horizon.

     (d)  Test  procedures—(1)  Summary  of the test. Surface soil  is
sieved and supplemented  with ground, dry alfalfa.  The  test substance, if
soluble, is added as a solution to moisten the soil, or is added  in a manner

that best simulates its anticipated mode of entry in nature. All soil samples
are then incubated in darkness at approximately 22  °C.  On  days 5 and
28 after introduction of the test substance,  samples are analyzed for NH3
and NOs content to establish ammonification and nitrification values, re-
spectively, and for CCh efflux as an indication of microbial respiration.

     (2) Application of test substance, (i) Deionized or glass-distilled
water should  be  used in making stock solutions  of  a water-soluble test
substance.  Sufficient quantities of each concentration should  be made  as
needed to minimize storage time and disposal volume. A constant volume
of the stock solution should be added at the beginning of the  test to each
soil sample designed to receive the test substance.

     (ii) A test substance that is insoluble in water, but which can be sus-
pended  in  an aqueous  solution by a carrier, should  be added, with the
carrier, to those soil samples designated to  receive the test substance. The
carrier should be soluble in water, nontoxic  to microbial life at the con-
centration applied,  and used in the minimum amount  required to dissolve
or suspend the test substance.  There are preferred carriers; however,  ace-
tone, gum arabic, ethanol, and others have been used extensively in testing
herbicides, plant growth regulators,  fungicides, and  other chemical sub-
stances that affect plants. Any such carrier  may be used for this test, pro-
viding it neither enhances nor inhibits  the  activities of the soil microbes.
Carrier controls should be included in the experimental design and tested
simultaneously with the test substance.

     (iii) A water-insoluble test substance  for which  no nontoxic, water-
soluble carrier is available  should be dissolved in an appropriate  volatile
solvent. The solution should be mixed with the ground alfalfa  soil supple-
ment, then placed in a rotary  vacuum apparatus  and  evaporated,  leaving
a uniform  coating  of the test  substance on the alfalfa. A portion of the
alfalfa should be weighed and the test substance should be extracted with
the same organic solvent. Then the test substance should be assayed before
the alfalfa  is mixed with the soil in the test containers. Solvent controls
(i.e., alfalfa treated only with  solvent) should be included in the experi-
mental design and tested simultaneously with the test substance.

     (iv) If the test substance is not readily soluble in water or in another
commonly-used carrier, and is known to be applied or  transported in nature
directly to the soil as a previously prepared  liquid or powder, it should
be mixed, in its liquid or dry form, directly into the soil samples. Mixing
must be  thorough,  however, to ensure equal distribution  of the test sub-
stance throughout each test sample.

     (3) Range-finding test, (i) A range-finding test  should be conducted
to establish (A) if definitive testing is necessary and (B) the concentrations
of test substance to be used in the  definitive test.

     (ii) If the maximum concentration of test substance to which the soil
microbial community is likely to be exposed in nature can be predicted,
soil  samples should be  treated with concentrations that are 0.1, 1, and 10
times the anticipated environmental exposure concentration. On days 5 and
28 after introduction of the test substance, the effects of treatment should
be assessed as the  CCh efflux rate and the NOs and NH3 concentrations
per gram of dry soil in treated samples, relative to untreated controls and,
if applicable, carrier controls, and to values in freshly sieved (pretreatment)
soil. Should reasonable predictions of anticipated environmental exposure
concentrations not be possible, soil samples should be exposed to a series
of widely-spaced concentrations  (e.g.,  1,  10, 100,  1,000, 10,000 (ig/g) of
the test substance. In general, the highest concentration in the series should
not be less than 1,000 |ig/g, although for water-soluble test  substances,
it is  recommended that  levels not exceed 50 percent of the saturation con-
centration.  As before, CO2 efflux and NOs and NH3 concentrations should
be compared with controls.

     (iii) The test should consist of exposing at least two  samples of soil
from the same source to each concentration of test substance and to each
control, with the exception of the controls for which one sample will suf-
fice. To be appropriate for this guideline, a soil should possess a pH of
4 to 8, an  organic  matter content between 1 and 8 percent, a cation ex-
change capacity  greater than 7  meq/100 g, and  consist of less than 70
percent sand. Soils to which fertilizer or pesticide(s)  have been applied
within the  past 24  months should be  avoided. Soil collections should be
restricted to the surface soil. Large  objects should be removed manually,
and  the remaining  soil allowed  to  air-dry until sievable  (approximately
12 percent water content), after which it is passed through a 2-mm mesh
screen. For each sample, an amount of soil equivalent to approximately
50 g oven-dry weight should be  placed in an inert container. Widemouth
jars (for example, glass  canning, 0.5 pint or 110-mL capacity) are adequate
for this purpose.  At least one of these  samples,  considered to be the con-
trol,  should be extracted immediately to determine NH3 and NOs content
(see  paragraph (d)(4)(vii) of this guideline). Alfalfa, dried and ground to
pass through a 0.6-mm mesh screen, should be added (0.3 g)  to each re-
maining sample and the sample  should be thoroughly mixed. Water con-
tent  of the soil should be adjusted to approximately  10  kPa by  adding
distilled water  containing the desired concentration  of test substance (in
carrier, if necessary). If insoluble in both water and commonly used car-
riers, the test substance should be mixed into the  soil as  a solid and the
appropriate amount of water added subsequently. The test  containers may
be covered with  0.13 (im (0.5 mil) polyethylene to minimize  water loss,
yet permit  gas exchange, or left open and watered to their  original weight
every 7 days. Regardless, the test substance should be applied only during
the original watering.

     (iv) Controls should receive an equal volume of water without the
test substance. If a carrier solvent is required to dissolve or suspend the
test substance, a carrier control (i.e., solvent in water without the test sub-
stance)  should also be included.  Should the  test substance be a powder
that is mixed directly into soil and subsequently moistened with distilled
water, the control should receive an equal volume of such water  only.
All samples should be incubated in darkness  at 22 °C (or at the tempera-
ture to which the microorganisms are most accustomed in their soil  envi-

     (v) Of the soil samples prepared for each concentration of test sub-
stance or  control, one sample should be assayed after 5 days of exposure
to the test substance for NH3 and NOs content, and then discarded.  A
second  sample should be analyzed the  same day for CCh evolution and
then reincubated  (dark, 22 °C). On day 28, after exposure to the test sub-
stance, all remaining (reincubated) samples should be assayed first for CO2
efflux and then again  for nitrogen content.

     (vi) The  test substance should be chemically stable in distilled water
or in any chemical substance used as a carrier.

     (vii)  No  replicates are required, and nominal concentrations  are ac-
ceptable unless definitive testing is not required.

     (viii) Definitive  testing is not necessary if the highest concentration
of test substance  tested (but not less  than 1,000 |ig/g) results in less than
a 50 percent reduction of ammonification, nitrification, and CO2 evolution;
or if that concentration representing the analytical detection limit (if tested)
results in greater than  a  50 percent  reduction  of NHs  and NOs  content
of the soil and of CO2 generation.

     (4) Definitive test, (i) The purpose of the definitive test is to deter-
mine  whether the test substance is toxic to the community of microorga-
nisms residing in a particular soil  and, if so, to delineate its concentration-
response curves and EC50s for each of three variables (CO2  evolution and
NHs and NOs soil content) that indicate the capacity of the  soil microbial
community to decompose organic  matter and release plant nutrients.

     (ii) Preparations for the test should be made  as  described  for the
range-finding test (see  paragraphs (d)(3)(iii)  through  (d)(3)(v)  of this
guideline) except that more samples  of each soil  source to be tested are
required. As before, at least two series of soil samples should be prepared
for each concentration; one series to be analyzed 5 days after exposure,
the other to be analyzed on the 28th day after exposure. Series replicates
of at least  five  concentrations  of test  substance, exclusive of controls,
should be used for each of the  two series.  For each soil source tested,
the concentration range should be  selected to define, as closely as possible,
the concentration-response curve between the ECio and ECgo for each vari-

     (iii) Every test should include controls consisting of the same distilled
water, soil,  and alfalfa supplement used in the treated soil samples, that
none of the  test substance is added. Environmental conditions should like-
wise  be  the same.  If a carrier is needed to dissolve or  suspend the test
substance, a carrier control should also be included.

     (iv) Test containers  may be covered with  polyethylene  film  (0.13-
(im; 0.5-mil) to prevent water loss. Control containers should be handled
identically to  the test containers except that none of the test substance
is added.

     (v) The definitive test consists of testing soil (containing the natural
microbial population) from a particular source with the test substance (see
paragraph (d)(3)(iii) of this guideline).  For a particular test substance, a
test is the exposure of the  selected soil to two identical series of five con-
centrations of the test substance in a minimum of five replicate containers
per concentration, and includes appropriate controls, with analyses of CC>2,
NHs, and NOs  on  the  5th and  28th  days after exposure  to the test sub-

     (vi) To measure CCh efflux, each container of test substance and con-
trol  container  is closed with a two-hole stopper fitted with Teflon tubes
and  twistcock  connectors  for attachment to the measurement apparatus.
The apparatus (see  Figure  1. following)  should deliver a stream of humid,
CCh-free air to the test system, and the effluent air should be dried, diluted,
and  delivered  for infrared gas analysis (IRGA).  The period of incubation
should be adjusted to match the CCh efflux rate with the detection capabil-
ity  of the IRGA, and may vary from 1 to 77 h.  In lieu of IRGA, CCh
may be trapped in  a hydroxide solution  and titrated, or measured by gas

ING CCh Evolution from Soil Samples



     (vii) Accumulation of inorganic  nitrogen is  measured by  extracting
each soil sample with 80 mL of 1 N KC1. After adding KC1 and shaking
each container by hand to suspend the soil, sample containers  should be
placed on a rotary  shaker at high  speed for 1 h, then shaken  again by
hand to resuspend the soil. Samples should be filtered (Whatman 42 low-
nitrate filter paper)  and the extract (filtrate) should be analyzed for NOs
and NH3.  Being rapid and precise,  standard Autotechnicon analysis tech-
niques are recommended. Acceptable alternative methods are  available,

     (viii) The assignment of soil containers to test substance concentra-
tions should be random. In  addition,  placement of the containers in the
incubation chamber should be randomized.

     (ix) Temperature in the incubation chamber should be monitored con-

     (5) Analytical  measurements-(i) Chemical. For readily  aqueous-
soluble test substances, stock solutions of the test substance should be di-
luted with glass-distilled or  deionized water to obtain the test  solutions.
Standard analytical methods  should be used to establish concentrations of
these solutions and should be validated before beginning the test. An ana-
lytical method is not acceptable if likely degradation products of the test
substance, such as hydrolysis and oxidation products, give positive or neg-
ative interference. The pH of these  solutions should also be measured be-
fore  use.

     (ii) Numerical. CC>2 efflux rates (micrograms per gram of dry soil/
h) and NOs and NHs concentrations  (micrograms per gram of dry soil)
in treated samples should be determined and compared with values ob-
tained from untreated controls,  carrier controls (if a carrier is used), and
from the freshly sieved, pretreatment soil. The significance of differences
between means may be established using Duncan's Multiple Range Test.
Means and standard deviations  should be plotted for each treated sample

and each control. Appropriate statistical analyses  should provide a good-
ness-of-fit determination for the concentration-response curves.

    (e) Test conditions—(1) Test species.  No particular species of test
organisms are recommended for use in this test due to the emphasis placed
on maintaining the natural state of the soil samples and their resident popu-
lations of microorganisms.  The test  organisms are, therefore, those that
occur naturally in the soil, and no others are to be introduced.

    (2) Facilities—(i) Apparatus. (A) Test chambers  should provide ade-
quate space and controls necessary to incubate numerous  soil samples in
total darkness at a constant temperature for prolonged periods of time.
Chambers should be designed to prevent escape  of internal  air into the
external environment other than  through appropriate filtering material or
media to prevent contamination of the external environment with the test

    (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 test and wash
solutions containing the test substance at the end of each test, and any
bench covering, lab clothing, or other  contaminated materials.

    (ii) Containers. For each test, at least 60 to  70 soil containers  (two
series of five per concentration of test  substance, two  series  of five for
the control,  and two series of five if a carrier control is necessary) should
be used. In addition, soil  to be  extracted immediately  as the  control is
most easily  handled in  an identical container. All  containers used in  each
experiment should be of equal size and volume, possess the same configu-
ration, and should be made of the same inert-material.

    (iii) Cleaning  and sterilization. (A) Soil containers and test solution
storage containers should be cleaned  before use. All equipment should be
washed according to good standard laboratory procedures to  remove any
residues remaining from manufacturing or prior use. A  dichromate solution
should not be used for cleaning containers.

    (B) If cleaning and rinsing of previously used soil  containers has been
thorough,  the effects of any microorganisms remaining on the interior sur-
face of the  containers  should be  insignificant in the presence of the new
test soil. Sterilization should not be necessary, but  is considered an accept-
able option.

    (C) Soil treated with the test substance and solvent control soil should
be discarded at  the end of the experiment.  Disposal  should conform to
applicable Federal regulations.

    (3) Test parameters. Environmental conditions should be maintained
as follows:

     (i) Constant incubation temperature of 22 °C (or that temperature to
which the microorganisms are most accustomed in nature).

     (ii)  Total  darkness  during  incubation  to prevent photosynthesis by
algae or the growth of moss.

     (f) Reporting. The final report should include, but not necessarily
be limited 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.

     (5) Stability of the test and, if used, control substances under the con-
ditions of administration.

     (6) A description of the methods used, which should include the fol-

     (i) Description of environmental conditions, including type and size
of incubation chamber and temperature used.

     (ii)  Description of test  diluent/solvent if other than distilled water,
e.g., if carrier is required.

     (iii) Description of experimental design and/or arrangement of equip-
ment, including a diagram, if complex.

     (iv) Methods used to determine the  placement of soil containers in
the incubation chamber and the  assignment of test  concentrations to con-
tainers to ensure randomization of exposure.

     (v) Frequency and methods of adding water to soil containers  during
the test period.

     (7)  A description of the test system used, including the  source  of
the test soil, the type of ecosystem from which it was removed, its chemi-
cal and physical characteristics  (mechanical analysis), and any available
geological information including soil type (classification).

     (8) A description of the  amount of soil tested per concentration, num-
ber of replicates, carrier (if any),  and incubation periods.


     (9)  The concentration of the test substance per unit dry weight of
test  soil  when the test substance is dissolved in water, solubilized with
a carrier, or coated on the alfalfa supplement and/or mixed  into the soil.

     (10) pH of the test solution applied to the soil samples.  The reported
results should include:

     (i) The results of the range-finding test expressed as  micrograms of
     evolved  per  gram of dry soil  per hour,  and micrograms of each of
     and NOs present per gram of dry soil, in treated and untreated sam-
ples. If the range-finding test indicated that the highest concentration of
the test substance tested (but not less than  1,000 |ig/g) had no effect on
the test system, report the results by soil source and type and state  that
the test  substance  has a  low potential for  adversely affecting microbial
functions in such soils. If the range-finding test indicated a greater than
50 percent reduction  of the  endpoints of the test at a concentration of
the test substance that represents the analytical detection limit (if tested),
report  the results by soil source and type and state  that the test substance
is  toxic to microbial life in such soils at concentrations at  or below the
analytical detection limit used in this study.

     (ii)  For the definitive test, the soil  source and  type, concentrations
of test substance used (micrograms per gram  dry  soil), and data for the
same variables used in the range-finding  test (see  paragraph (f)(l)(x)(A)
of this  guideline) should be reported.

     (11) A description of all circumstances  that may have affected the
quality or integrity of the data.

     (12) 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.

     (13)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., X test, and EC50s.

     (14) The signed and dated reports of each of the individual scientists
or other  professionals 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.

     (15) The locations where all specimens, raw data, and the final report
are stored.

     (16) The statement prepared and signed by the  quality assurance unit.