United States Prevention, Pesticides EPA712-C-98-088
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3300
Soil Biodegradation
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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
(7U.S.C. I36,etseq.).
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 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 835.3300 Soil biodegradation.
(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 materials used in developing this har-
monized OPPTS test guideline are the OPPT guideline under 40 CFR
796.3400 and OECD 304 A Inherent Biodegradability in Soil.
(b) Introductory information—(1) Prerequisites. 14C-labeled mate-
rial is required.
(2) Guidance information. Information on the toxicity of the test
compound is useful for the interpretation of the data obtained. The con-
centration of the test compound can then be adapted to this information.
(3) Qualifying statements. The test is applicable to volatile or non-
volatile, soluble or insoluble compounds which are not inhibitory to micro-
organisms. The mineralization rate refers to the labeled carbonation only.
Therefore, the location of the labelling within the structure and the speci-
ficity of the label need careful consideration.
(4) Recommendations, (i) The results obtained using the basic min-
eralization test may be supported by determination of the evaporation rate
of the parent compound and some of possible volatile metabolites and by
determination of soil extractable and nonextractable residues. Both op-
tional tests are described in this test guideline.
(ii) Sometimes it is recommended that information about chemical
degradation under anaerobic conditions be obtained. Therefore, in accord-
ance with the description below, the biometer flask filled with the soil
sample (preconditioning is not necessary), is flooded with water (2-3 cm
layer) to protect against leakage, evacuated and flushed with nitrogen sev-
eral times. Degradation may be evaluated by means of measurements of
methane gas and analysis of both water and soil for 14C-content.
(5) Standard documents. This test guideline is based on the method
cited in paragraph (e)(l) of this guideline.
(c) Method—(1) Introduction, purpose, scope, relevance, applica-
tion and limits of test, (i) The method described in this test guideline
is designed for the evaluation of the mineralization rate of a 14C-labeled
compound in soil. The method is applicable to volatile or nonvolatile, solu-
ble or insoluble compounds which are not inhibitory to microorganisms.
(ii) Definitions and units. Soil is a mixture of mineral and organic
chemical constituents, the latter containing compounds of high carbon and
nitrogen content and of high molecular weights, animated by small (mostly
micro-) organisms. Soil may be handled in two states: Undisturbed, as
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it has grown with time, in characteristic layers of a variety of soil types,
or disturbed, as it is usually sampled by digging and used in the test de-
scribed here.
Mineralization (in the context of this guideline) means extensive deg-
radation of a molecule during which a labelled carbon atom is oxidized
quantitatively with release of the appropriate amount of14 CO2.
(iii) Reference substances. In some cases when investigating a new
substance reference substances may be useful; however, reference sub-
stances cannot yet be recommended. Reference substances need not be
employed in all cases when investigating a new substance. They may pri-
marily be used so that calibration of the method may be performed from
time to time and to permit comparison of results when another method
is employed.
(iv) Principle of the test method—(A) Basic test. (7) A small sam-
ple of soil is treated with the 14 C-labeled test chemical in a biometer flask
apparatus. Release of 14 CCh from the test chemical is measured by means
of alkali absorption and liquid scintillation counting.
(2) Optional experiments include the following tests.
(B) Evaporation test. When testing chemicals of a vapor pressure
higher than 0.0133 Pa, a polyurethane foam plug is placed into the biom-
eter flask apparatus to absorb the labelled volatile part of the parent
compound and volatile metabolites for liquid scintillation counting.
(C) Residue test. At the point of 50 percent mineralization the test
soil may be extracted. The extractable portion of the compound, and its
metabolites remaining in the soil, may be determined by liquid scintillation
counting. Furthermore, data on the bound residue part may be obtained
by measuring the 14 CO2 released after combustion of the soil.
(v) Quality criteria—(A) Reproducibility. Reproducibility is good
if standard conditions, especially preconditioning of the soil, are strictly
observed.
(B) Sensitivity. The evaluation of sensitivity is not relevant because
a moderate amount as 37-185 kBq (=1-5 (iCi) of 14 C-labeled test chemi-
cals is used for each experiment.
(C) Specificity. The method is only applicable if 14 C-labeled test
chemicals are available. The specificity is very good.
(D) Possibility of standardization. This procedure is standardized
to a limited extent. The limitation is related to the difficulty of standardiza-
tion of soil samples between laboratories.
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(2) Description of the test procedure—(i) Preparations—(A)
Equipment. (7) Liquid scintillation counter.
(2) Oxidizer for combustion of radioactive material.
(3) Ultrasonic bath, 500 mL.
(4) Glassware: 250 mL Erlenmeyer flasks fused to 50 mL round bot-
tom tubes (biometer flasks, see Figure 1); 25 mL syringes (e.g. Luer-Lok);
15 gauge syringe needle, 15 cm in length; 100 \\L syringes (e.g. Hamilton);
25 mL graduated cylinders with stopper; 1 mL pipets; Soxhlet extraction
apparatus; scintillation vials; polyurethane plugs, 30 mm diameter, 30 mm
length, density 16 kg/m3
(B) Reagents—(7) Test substance: 14 C-labeled compounds are dis-
solved in water or acetone to give radioactivity of 37-185 KBq
(=1-5 (iCi)/100 (iL. Using unlabeled material this solution is made up to
the required concentration (e.g. 0.5 mg/100 \\L = 10 mg/kg soil, or depend-
ing on the toxicity of the substance).
(2) Chemicals. (/) KOH, analytical grade, 0.1 N solution.
(if) Acetone, analytical grade.
(fff) Methanol, analytical grade (for optional tests).
(fv) w-Hexane, analytical grade (for optional test).
(v) Ascarite (A.H. Thomas Co. Philadelphia or equivalent).
(vf) Scintillation cocktail.
(3) Soil. (/) Alfisol: pH between 5.5 and 6.5 organic carbon content
between 1 and 1.5 percent clay content (i.e. particles <0.002 mm in diame-
ter) between 10 and 20 percent cation exchange capacity between 10 and
15 mval.
(//) Spodosol: pH between 4.0 and 5.0 organic carbon content between
1.5 and 3.5 percent clay content <10 percent cation exchange capacity
< 10 mval.
(fff) Entisol: pH between 6.6 and 8.0 organic carbon content between
1 and 4 percent clay content between 11 and 25 percent cation exchange
capacity > 10 mval.
In special cases it is recommended that two additional soils be used: One
with high silt-fraction (diameter between 0.002 and 0.063 mm) content,
the other with a high clay content (30 percent). Air dried test soil stored
at +4 °C is remoisturized to 40 per cent maximum water capacity. After
incubation for 2 weeks at 22 + 2 °C in the dark it is ready for the experi-
ments.
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(ii) Test conditions—(A) Test temperature. During the whole test
period the flasks are incubated in the dark at 22 ±2 °C.
(B) Soil characterization data. (7) for determination of the pH value
of the soil for selecting the soil type, 10 g air-dried soil is suspended
in25mL0.01MCaCl2.
(2) After standing overnight, the sample is disturbed once more and
measured in a potentiometric apparatus with a 0.1 M KC1 electrode. Imme-
diately before the measurement the instrument must be calibrated with two
standard solutions within the measuring range of the sample values ex-
pected.
(3) For determination of the organic carbon content of the soil for
selecting the soil type, 1.0 g air-dried soil is heated with 15 mL
2M K2Kicr2O7 and 20 ml H2SO4 (analytical reagent, pc = 1.84 g/cm3)
at 145-155 ° C for 15 min. After cooling to room temperature sample
volume is made up to 150 mL with distilled water. A 20 mL aliquot is
measured spectrophotometrically, after centrifuging, in a 1 cm cuvet at
590 nm compared to distilled water. The self-destroying property of the
K2Cr2O7 reagent must be determined by two blank samples. Calculation
is conducted using the following equation:
1,000 x v x E2(EX - OC2 x c)
C =
e x EI x (a, - OC2 x F)
where
C = carbon content (percent)
V = gross volume (mL)
EI = equivalent weight of Cr2O3 (25.332)
E2 = equivalent weight of carbon (3.0028)
Ex = extinction at 590 nm and 1 cm layer thickness
F = factor for calculating K^C^O? from C^Os
c = concentration of Cr (g) per 100 mL (= 1.9356)
e = sample weight (mg)
oci = extinction coefficient of Cr(III); oci is an average value from five single
determinations for the calibration curve, each obtained by division of EX
by the amount of Cr2O3 (g)
OC2 = extinction coefficient of Cr(VI); a2 is an average value from two single
estimations, each obtained by division of Ex by the respective amounts of
K2Cr2O7.
(4) To determine particle size of the soil for selecting the soil type,
10.0 g air-dried soil is reacted with 100 mL H2O2 (15 percent W/V) for
15 h, heated until CO2 evolution is complete. The suspension is left to
stand overnight with 25 mL 0.4 N Na4P2Oy, after which water is added
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to make it up to 250 mL and the solution is sieved through a mesh of
0.2 mm width. The portion <0.2 mm is fractionated further by sieving.
The smaller particles (silty fractions) are fractionated by homogenous par-
titioning of the particles in the aqueous medium, which is made up to
1,000 mL with water in an elutriating cylinder.
(5) Portions (10 mL) are removed by pipet from various heights of
the cylinder after different sedimentation times; measurement of the dry
weights of the suspended material in these portions yields the particle com-
position according to the following scheme:
Particle fraction
diameter (mm)
<0.002 ....
<0.0063
<0.02
<0063
20
1h 33min 49sec
9min 19sec
59sec
Dipping depth (cm)
15 10
7h 45min
1h 10min 52sec 46min 55sec
6min 59sec 4min 39sec
42sec
5
3h 52min
23min 27sec
2min 20sec
(6) For determination of the cation exchange capacity of the soil, in
order to select the soil type, a glass column 15 cm in length and 30 mm
i. d. is reduced in diameter at one end like a funnel. This side is stuffed
with glass wool. About 1 cm quartz sand is spread on the wool, followed
by 10.0 g air-dried test soil, which is in turn covered by about 1 cm of
quartz sand. Forty milliliters of a mixed solution consisting of 100 g tri-
ethanolamine in 2 L water (adjusted to ph 8.1 with HC1) plus 100 g
BaCL: 2H2O in 2 L) is poured over these layers. After 1 h the solution
is collected in a 250 mL Erlenmyer flask. The procedure is repeated. In
addition, 40 mL of a solution of 25 g BaCL: 2H2O in 1 L is poured onto
the column.
(7) This solution is collected after standing overnight and the column
is washed with 100 mL water. The combined eluates are titrated against
HC1 (bromocresol green plus methyl red as indicators) to measure H+,
Ca2+, K+, Na+. The column is leached in a similar mannner with 200
mL of 20 g MgCl2 6H2O in 1 L water for the determination of Ba2+. This
cation is determined by flame absorption spectrophotometry. The cation
exchange capacity is expressed as the sum of all the cation equivalents
sorbed by 100 g soil.
(iii) Performance of test—(A) Basic test. (7) Fifty grams of soil
(dry weight basis) is placed into each Erlenmeyer part (H) of the biometer
flask (see the following Figure 1). The radioactive test solution (100 (iL)
is added in 50 drops over the whole soil surface (I) of each flask. The
soil is carefully mixed with a Pasteur pipet (from which the lower part
is cut off) and left in the flask.
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FIGURE 1—TEST FLASK
(2) In addition, an equivalent volume of test solution is placed in
a 100 mL volumetric flask for direct determination of the added radioactiv-
ity.
The biometer flask is closed with a Teflon-coated silicon rubber
stopper through which an Ascarite filter (F) is inserted. The filter (F) is
provided with a stopper and stopcock (G). The side tube (C) is sealed
with a teflon stopper pierced by a 15-gauge needle (B), 15 cm long. The
needle (B) is capped by a silicone rubber stopper (A), and its tip at (D)
is covered with a short length of silicone tubing that remains in contact
with the base of the side tube (C).
(4) The unit is charged by injecting 10 mL of alkali solution into
the side tube (C) in the following manner: the small stopper (A) is replaced
by a calibrated Luer Lok syringe containing 0. 1 N KOH; the filter stopper
on (F) is removed and the stopcock (G) is opened; the alkali solution is
introduced through the needle (B) into the side tube (C); the stopcock
is closed; the syringe is removed; the small stopper (A) and filter stopper
on (F) are returned to their initial positions. The 14 C-carbon dioxide pro-
duced is absorbed by the alkali.
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(5) To recover the 14 C02-loaded alkali for liquid scintillation analysis,
the procedure for charging each parallel unit at increasing time intervals
after start of the experiment is performed in reverse order. Thereafter the
side tube (C) is rinsed with 5 mL alkali. Before recharging the side tube
(C) fresh alkali, 3 volumes of 25 mL air are sucked through the system
with the empty syringe to maintain the soil in an aerobic condition. A
1 mL aliquot of the alkali solution is taken for liquid scintillation counting.
(6) Experiment duration times of 1, 2, 4, 8, 16, 32 and—if nec-
essary—64 days should be chosen for measurement. The test requires par-
allel determinations. The 14CC>2 radioactivity recovered is plotted versus
time. This graph shows when to terminate the experiment. Incubation time
is sufficient, when a total of 50 per cent CCh expressed as 14C originally
applied can be measured. Incubation should be stopped after reaching 64
days, whether or not this value is obtained.
(B) Optional tests—(7) Estimation of evaporation. If the volatility
of a chemical is higher than 10 5 torr at 20 °C, it is recommended that
a 3 cm diameter polyurethane foam plug be introduced into the arm E
of the biometer flask. This plug absorbs the volatile parent compound as
well as volatile organic degradation products but does not absorb 14CC>2.
The plugs are extracted in a Soxhlet extraction apparatus with an w-hexane/
methanol mixture (1:4), and aliquots are taken for liquid scintillation
counting.
(2) Determination of soil-extractable and non-extractable resi-
dues. (/) In cases of relatively persistent chemicals (50 percent mineraliza-
tion in 10 days), further information concerning the soil-extractable radio-
activity (parent compound plus degradation products) and soil bound resi-
dues is recommended.
(if) For this purpose two further biometer flasks in addition to the
four others must be prepared. At the point of 50 (or x-) percent mineraliza-
tion in the basic test, the soil in the two separate biometer flasks is ex-
tracted with 100 mL acetone (5 min ultrasonic treatment) followed by an
extraction with methanol in the same manner. Aliquots of the combined
extracts are taken for liquid scintillation counting. Other extract portions
may be used—if necessary—for further identification studies.
(///) Aliquots of the air dried soil are combusted to 14CC>2 and meas-
ured by liquid scintillation counting to determine the soil bound residues.
(d) Data and reporting—(1) Treatment of results—(i) Basic test.
Radioactivity values for 14CC>2 (average of 4 parallel experiments) ob-
tained from the alkali solution after 1, 2, 4, 8, 16, 32 and 64 days are
expressed as the percentage of test chemical (radioactivity) initially applied
and are plotted in a graph versus time. The time at which 50 percent of
the radioactivity is recovered as 14CC>2 is considered to be the "50 percent
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mineralization'' level. If this level has not been reached by the 64th day,
the data at this time are taken and expressed as X-percent-mineralization.
(ii) Evaporation test. The radioactivity of vaporized (and trapped)
original compound plus degradation products at the point of 50-
(or X-) percent mineralization is extracted, measured and interpreted as
the percentage of volatilization at the point of 50- (or X-) percent-min-
eralization.
(iii) Residue test. Radioactivity values for extractable and non-ex-
tractable residues of the parent compound plus degradation products ob-
tained after the extraction procedure of the soil at the point of 50-
(or X-) percent-mineralization are given.
(2) Test report. The report of the degradability of a test chemical
must include:
(i) Name of the test chemical, formula.
(ii) Amount applied, if not standard.
(iii) Exact characteristic data of the soil used.
(iv) Dates of the performance of the measurements.
(3) Interpretation and evaluation of results. The results are artifi-
cial because they are obtained with disturbed soil. However, since stand-
ardized soils are used, the test data are intercomparable and enable the
experimentator to group relatively the chemicals tested within one scale
for this property.
(e) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Bartha, R. and Pramer D. Soil Science 100:68-70 (1965).
(2) Soil Survey Staff. U.S. Department of Agriculture. Soil Taxonomy
Handbook No. 436. Washington, DC (1975).
(3) Butler, B.E. Soil Classification for Soil Survey Oxford (980) p.
129.
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