United States Prevention, Pesticides EPA712-C-98-098
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.5154
Anaerobic
Biodegradation in the
Subsurface
-------
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."
-------
OPPTS 835.5154 Anaerobic biodegradation in the subsurface
(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
795.54 Anaerobic Microbiological Transformation Rate Data for Chemi-
cals in the Subsurface Environment.
(b) Introduction. (1) This guideline describes laboratory methods for
developing anaerobic microbiological transformation rate data for organic
chemicals in subsurface materials. The method is based on a time-tiered
approach. For chemicals that are degraded rapidly, only a portion (for ex-
ample, the 0-, 4-, and 8-week sampling periods) of the test will have to
be completed; however, for slowly degrading chemicals, the entire test
may have to be performed (64 weeks). The data will be used to calculate
degradation rate constants for each tested chemical over a range of envi-
ronmental conditions. The rate constants obtained from testing will be inte-
grated into algorithms to assess the fate of organic chemicals leaching into
ground water from waste management facilities.
(2) Anaerobic transformations are evaluated under methanogenic and
sulfur-reducing conditions. Aerobic biodegradation was not included in the
modeling analysis for two reasons:
(i) Aerobic biodegradation would be limited by the concentration of
oxygen in ground water. In the laboratory, oxygen would probably not
be limiting, and the resulting degradation rates obtained would possibly
be overestimations of actual subsurface degradation rates.
(ii) Aerobic degradation would only occur at the leading edge of a
contaminant plume where dispersion and other processes dilute the plume
with oxygenated water, as stated in Wilson et al. (1985), in paragraph
(d)(24) of this guideline.
(3) The anaerobic transformation of chemicals in selected subsurface
samples should be estimated from subsurface microcosm studies using
methods adapted from procedures recently reported by Wilson et al.
(1986), in paragraph (e)(25) of this guideline. These procedures should
be used to determine the length of the adaptation period (time interval
before detectable degradation of the chemical can be observed) and the
half-life of the chemical following the adaptation period. Supporting lab-
oratory methods should be used to measure the levels of residual test
chemical, intermediate degradation products, biomass, and other physical-
chemical parameters.
-------
(c) Laboratory procedures—(1) Identification of subsurface sam-
pling sites, collection of subsurface materials, and transportation and
storage of subsurface materials, (i) A minimum of six subsurface sam-
pling sites should be identified on the basis of two temperatures and three
pH values. Three of the sites should have annual average temperatures
near 10 °C, and three of the sites should have temperatures near 20 °C.
These values are chosen to represent the high and low temperatures com-
monly observed in aquifers and are one standard deviation on either side
of the mean temperature of 15 °C. Generally, low temperature sites are
located in northern latitude areas of the United States, and high tempera-
tures correspond to southern latitude areas.
(ii) Acidic (pH 4.5 to 6.0), neutral (pH 6.5 to 7.5), and alkaline (pH
8.0 to 9.5) sites should be selected for each temperature range. These
ranges of pH values for ground waters are selected to estimate the effect
of pH on microbial degradation capacity and to examine the effect of
chemical form on the degradation of chemicals having dissociable hydro-
gen (i.e., degradation of the protonated and unprotonated forms of the
chemical). Ground waters at all sites should have dissolved-C^ levels at
<0.1 mg/L and sulfate concentrations at < 10 mg/L.
(iii) Samples of subsurface materials should be collected in a manner
that protects them from contamination from surface materials and main-
tains anaerobic conditions. An appropriate procedure has been reported
by Wilson et al. (1983), in paragraph (e)(26) of this guideline. First, a
bore hole is drilled to the desired depth with an auger. Then the auger
is removed and the sample taken with a wireline piston core barrel, as
reported by Zapico et al. (1987), in paragraph (e)(14) of this guideline.
The core barrel is immediately transferred to an anerobic chamber, which
is filled and continually purged with nitrogen gas, and all further manipula-
tions are performed in the chamber. Using aseptic procedures, up to
5 cm of the core is extruded, then broken off to produce an uncontaminated
face. A sterile paring device is then installed, and the middle 30 to
35 cm of the core is extruded, paring away the outer 1.0 cm of core mate-
rial. As a result, the material that had been in contact with the core barrel,
and thus might be contaminated with surface microorganisms, is discarded.
Modifications of this technique can be used for samples obtained from
deep coring devices when auger equipment is insufficient because of the
depth of the aquifer. Subsurface material should be stored under nitrogen
gas and on ice and should be used in microcosm studies within 7 days
of collection.
(iv) Ground waters will be collected from the bore hole used to collect
subsurface materials. Ground waters will be pumped to the surface. The
bore hole should be purged with argon before pumping begins. The pump-
ing mechanism should be flushed with enough ground water to ensure
that a representative ground water sample is obtained. This flushing proc-
ess generally requires a volume equal to 3 to 10 times the volume of
-------
water in the bore hole. Once flushing is complete, ground water samples
should be collected, and stored under nitrogen and on ice for transport
back to the laboratory. Ground waters should be sterilized by filtration
through 0.22 (im membranes on-site in a portable anaerobic chamber
which has been filled and continually purged with nitrogen gas. The sterile
water should be stored under nitrogen and on ice, and should be used
in microcosm studies within 7 days of collection.
(v) Two samples should be collected from each of the six sites. Each
core sample should be assayed for test chemical degradation and analyzed
for biomass (heterotrophic, sulfate-reducing, and methanogenic) and phys-
ical-chemical parameters (pH, cation exchange capacity, total organic car-
bon, percent base saturation, percent silt, percent sand, percent clay, redox
potential, percent ash-free dry weight). Each corresponding ground water
sample will be analyzed for pH, dissolved oxygen, dissolved organic car-
bon, nutrients (sulfate, phosphate, nitrate), conductivity, and temperature.
(2) Anaerobic microcosm assay, (i) Microcosms should consist of
160-mL serum bottles which have been filled completely with a slurry
of subsurface material and ground water (20 gm equivalent dry weight
(dried at 103 °C) solid to 80 mL ground water). One series of serum bot-
tles should be amended to a level of 200 mg/L sulfate added as sodium
sulfate to stimulate sulfate-reducing conditions. If the level of soluble sul-
fate falls below 50 mg/L at any sampling time, additional sulfate
(200 mg/L) should be added to all remaining sulfate-amended microcosms.
Soluble sulfate levels should be measured by the method of Watwood et
al. (1986), in paragraph (e)(23) of this guideline. A second series should
be left unamended to simulate methanogenic conditions. All manipulations
in preparing the microcosms should be performed aseptically under strict
anaerobic conditions, as described in Kaspar and Tiedje (1982) in para-
graph (e)(10) of this guideline, or other equivalent methods, and all equip-
ment in contact with the subsurface samples should be sterilized. Sterile
controls should be prepared by autoclaving the samples for a minimum
of 1 h on each of 3 consecutive days. Test chemical amendments should
be prepared in sterile nitrogen-purged ground water. Sparingly soluble and
volatile chemicals should be added to sterile, nitrogen-purged ground water
and then stirred overnight without a head space.
(ii) The active and control microcosms should be dosed with the test
chemical and 0.0002 percent (W/V) Resazurin as a redox indicator, and
then each unit should be immediately sealed with a Teflon-coated gray
butyl rubber septum and crimp seal. As stated previously, all manipulations
should be performed under strict anaerobic conditions, as described in
Kaspar and Tiedje (1982) in paragraph (e)(10) of this guideline, or other
equivalent methods. The microcosms should be stored in the dark at the
original in situ temperature. Active microcosms and control microcosms,
randomly selected from the sulfate-amended series and the unamended se-
ries, should be sacrificed and analyzed at 0, 4, 8, 16, 32, and 64 weeks
-------
for residual test chemical and the formation of degradation intermediates.
Once the residual level of the chemical drops below 5 percent of the initial
concentration, analysis of microcosms at subsequent time periods is not
required. The active microcosms and control microcosms from both series,
at weeks 0, 16, and 64 (or randomly selected from the remaining samples
the week following 95 percent degradation of the chemical, if less than
week 64) should also be analyzed for heterotrophic, sulfate-reducing, and
methanogenic bacteria.
(iii) Three concentrations of each chemical tested should be used. The
test chemical concentrations should range between a low level of 30 times
the health-based level and a level that equates to the chemical's solubility
(or to a level that causes inhibition of the test chemical's degradation).
(iv) Biomass measurements should be made for heterotrophic, sulfate-
reducing, and methanogenic bacteria. Biomass measurements have been
included to ensure comparability of results between samples of subsurface
materials. Degradation rates derived from sediment samples having signifi-
cantly high or low (student ' 't'' test, 90 percent level) bacterial populations
would not be considered in subsequent modeling efforts. Also, the ratio
of sulfate-reducing organisms to methanogenic organisms would be used
to determine if the dominant redox conditions were sulfate-reducing or
methanogenic. Anaerobic techniques described by Kaspar and Tiedje
(1982), cited in paragraph (e)(10) of this guideline, or other equivalent
methods, should be used.
(v) Heterotrophic bacterial concentrations should be measured by a
modification of the procedure developed by Molongoski and Klug (1976)
and Clark (1965), cited in paragraphs (e)(13) and (e)(6) of this guideline,
respectively. A 10-mL sample taken from the center of the appropriate
microcosm, which has been well mixed, should be aseptically transferred
to 100 mL of sterile dilution medium and agitated to suspend the orga-
nisms. Samples (10 mL volume) should then be transferred immediately
from the center of the suspension to a 90-mL sterile dilution medium blank
to give a 10 2 dilution; 10 mL should be similarly transferred to another
90-mL of sterile dilution medium to obtain a dilution of 10 3. This process
should be repeated to give a dilution series through at least 10 7. Only
the 10 -1 dilution need be prepared from control samples. The dilution se-
ries can be modified to include dilutions of greater than 10 7, if necessary,
and if sufficient sample is available. From the highest dilution, 0.1-mL
portions should be transferred to the surface of each of three dilute
tryptone glucose extract agar plates. The sample should be spread imme-
diately over the surface of the plates and the process should be repeated
for lower dilutions. Dilute tryptone glucose agar plates should be prepared
by combining 24.0 g tryptone glucose extract agar in 1 L of distilled water.
The mixture should be autoclaved, and 25 mL of the molten agar should
be transferred to Petri plates. Agar plates should be stored in an anaerobic
chamber for a minimum of 24 hours before use. The inoculated plates
-------
should be incubated in plastic bags in the glove box, or, if necessary, re-
moved and kept in anaerobic jars. After 14 days of incubation, the plates
should be examined and the total count per gram of dry sediment material
should be determined. If the plates from the most dilute sample show more
than 300 colonies, the dilution series was inadequate. In this case, all of
the plates should be discarded, and the process should be repeated with
greater dilutions, as appropriate.
(vi) Sulfate-reducing species should be enumerated by the MPN (most
probable number) technique as descibed in Pankhurst (1971) in paragraph
(e)(15) of this guideline, or other equivalent method. The dilution series
should be prepared as described for heterotrophic bacteria.
(vii) Methanogenic bacteria should be enumerated by the MPN tech-
nique as described by Jones et al. (1982) in paragraph (e)(9) of this guide-
line, or by another equivalent method. The dilution series should be pre-
pared as described for heterotrophic bacteria.
(3) Analytical measures of the loss of test chemical and intermedi-
ate degradation products, (i) The loss of test chemical should be quan-
tified by measuring the residual test chemical. The formation of degrada-
tion intermediates should be quantified in microcosm assays for test chemi-
cals that can potentially be transformed. Analysis for degradation inter-
mediates should be required when the level of test chemical has been re-
duced by more than 25 percent. Concentrations of the potential degradation
products 1,2-, 1,3-, and 1,4-dichlorobenzene, and 1,2,4,5-
tetrachlorobenzene should be measured in the appropriate microcosms used
to analyze the degradation of pentachlorobenzene. The concentration of
the potential degradation product dibromomethane should be measured in
the appropriate microcosms used to analyze the degradation of bromoform.
The potential degradation products methanethiol and chloromethane (meth-
yl chloride) should be measured in the appropriate microcosm used to ana-
lyze the degradation of trichloromethanethiol. The potential intermediate
products 1,2-, 1,3-, and 1,4-dichlorobenzene should be measured in the
appropriate microcosm used to analyze the degradation of 1,2,4,5-
tetrachlorobenzene.
(ii) Measurements of test chemical and intermediate degradation prod-
ucts will require organic analytical techniques tailored to the specific test
chemical and subsurface material being investigated. Several extraction
and purge-trap techniques are available for the recovery of residual test
chemicals and degradative intermediates from subsurface materials.
Unique analytical procedures would have to be developed or modified for
each test chemical and sediment. The following represent examples of such
techniques:
(A) Soxhlet extraction as described in Anderson et al. (1985), Bossart
et al. (1984), Eiceman et al. (1986), Grimalt et al. (1984), and Kjolholt
-------
(1985) under paragraphs (e)(2), (e)(3), (e)(7), (e)(8), and (e)(ll) of this
guideline, respectively.
(B) Shake flask method as described in Brunner et al. (1985), and
Russel and McDuffie (1983) in paragraphs (e) (4) and (16) of this guide-
line, respectively.
(C) Sonification as described in Schellenberg et al. (1984) in para-
graph (e)(17) of this guideline.
(D) Homogenization as described in Fowlie and Sulman (1986),
Lopez-Avila et al. (1983), Sims et al. (1982), Stott and Tabatabai (1985),
and U.S. EPA (1982) in paragraphs (e)(5), (e)(12), (e)(18), (e)(19), and
(e)(22) of this guideline, respectively.
(E) Purge-trap techniques have been described by Wilson et al. (1986)
in paragraph (e)(24) of this guideline.
(iii) These procedures can be readily coupled to gas chromatography
(GC) and high-pressure liquid chromatography (HPLC) procedures to
quantify the chemicals of interest. Whatever analytical procedure is se-
lected should follow Good Laboratory Practice Standards of 40 CFR part
792.
(4) Characterization of subsurface materials and ground waters.
(i) Subsurface materials should be classified, described, and characterized
as to soil type and physical and chemical properties using standard proce-
dures as described by the Soil Conservation Service (U.S. Department of
Agriculture, 1972 and 1975) under paragraphs (e)(20) and (e)(21) of this
guideline, or other equivalent methods. Ten parameters will be measured
as follows:
(A) Total organic carbon (TOC).
(B) pH.
(C) Cation exchange capacity.
(D) Percent base saturation.
(E) Percent silt.
(F) Percent sand.
(G) Percent clay.
(H) Redox potential.
(I) Percent ash-free dry weight.
(J) Texture.
-------
(ii) Ground water should be characterized for the following, by stand-
ard water and wastewater methods described by the American Public
Health Association (1985) in paragraph (e)(l) of this guideline, or other
equivalent methods:
(A) PH.
(B) Dissolved oxygen.
(C) Dissolved organic carbon.
(D) Nutrients including sulfate, phosphate, and nitrate.
(E) Conductivity.
(F) Temperature.
(iii) The properties of pH, dissolved oxygen, and temperature should
be measured at the site of collection. All other properties should be meas-
ured in the laboratory.
(d) Data to be reported to the Agency. Data should be reported
for the two subsurface samples and corresponding ground waters taken
from the six different sampling sites.
(1) The following should be reported for subsurface sediment sam-
ples:
(i) Levels of residual test chemicals (milligrams per gram of dry
weight) quantified in each of the randomly selected replicate microcosm
and sterile controls at the specific time periods identified under the anaer-
obic microcosm assay.
(ii) Numbers of heterotrophic, sulfate-reducing, and methanogenic
bacteria (colony forming units (CPU) or most probable number units
(MPNU) per gram dry weight) enumerated in each replicate microcosm
and sterile controls at the specific time periods identified under the anaer-
obic microcosm assay.
(iii) Levels of persistent degradation intermediates identified in micro-
cosm and sterile controls at the specific time periods identified under the
anaerobic microcosm assay.
(iv) Measured values for pH, cation exchange capacity (meq/100 gm
dry wt), percent base saturation, percent silt (percent dry weight), percent
sand (percent dry weight), percent clay (percent dry weight), redox poten-
tial (Eh, Standard Hydrogen Electrode), percent ash free dry weight (per-
cent dry weight), and a description of texture.
(2) For ground water samples, the analysis report should provide
measured values (in units specified) for:
-------
(i) pH.
(ii) Dissolved oxygen (in milligrams per liter).
(iii) Dissolved organic carbon (in milligrams per liter).
(iv) Nutrients including sulfate, phosphate, and nitrate (in milligrams
per liter).
(v) Conductivity (S, 25 °C).
(vi) Temperature (expressed as degrees Celsius).
(e) References. The following references should be consulted for ad-
ditional background information on this guideline,
(1) American Public Health Association, American Water Works As-
sociation, and Water Pollution Control Federation. Standard methods for
the examination of water and wastewater. 16th ed., A.E. Greenberg, R.R.
Trussel, and L.C. Clesceri (eds.), American Public Health Association,
Washington, DC (1985).
(2) Anderson, J.W. et al. Method verification for determination of
tetrachlorodibenzodioxine in soil. Chemosphere 14:1115-1126 (1985).
(3) Bossart, I. et al. Fate of hydrocarbons during oil sludge disposal
in soil. Applied and Environmental Microbiology 47:763-767 (1984).
(4) Brunner, W. et al. Enhanced biodegradation of polychlorinated
biphenyls in soil by analog enrichment and bacterial inoculation. Journal
of Environmental Quality 14:324-328 (1985).
(5) Fowlie, P.J.A., and T.L. Bulman. Extraction of anthracene and
benzo[a]pyrene from soil. Analytical Chemistry 58:721-723 (1986).
(6) Clark, F.E. Agar-plate method for total microbial count, p. 1460-
1466. In: C.A. Black (ed.), Methods of soil analysis. Part 2. Chemical
and Microbiological Properties. American Society of Agronomy, Inc.,
Madison WI (1965).
(7) Eiceman, G.A. et al. Depth profiles for hydrocarbons and
polycyclic aromatic hydrocarbons in soil beneath waste disposal pits from
natural gas production. Environmental Science and Technology. 20:508-
514(1986).
(8) Grimalt, J. et al. Analysis of hydrocarbons in aquatic sediments.
International Journal of Environmental Analytical Chemistry 18:183-194
(1984).
(9) Jones, J.G. et al. Factors affecting methanogenesis and associated
anaerobic processes in the sediments of a stratified eutrophic lake. Journal
of General Microbiology 128:1-11 (1982).
8
-------
(10) Kaspar, H.F., and J.M. Tiedje. Anaerobic bacteria and processes,
p. 989-1009. In: A.L. Page (ed.), Methods of Soil Analysis. Part 2. Chemi-
cal and Microbiological Properties. American Society of Agronomy, Madi-
son, WI (1982).
(11) Kjolholt, J. Determination of trace amounts of
organophosphorous pesticides and related compounds in soils and sedi-
ments using capillary gas chromatography and a nitrogen-phosphorous de-
tector. Journal of Chromatography 325:231-238 (1985).
(12) Lopez-Avila, V. et al. Determination of 51 priority organic com-
pounds after extraction from standard reference materials. Analytical
Chemistry 55:881-889 (1983).
(13) Molongoski, J.J. and M.J. Klug. Characterization of anaerobic
heterotrophic bacteria isolated from freshwater lake sediments. Applied En-
vironmental Microbiology 31:83-90 (1976).
(14) Zapico, M.M. et al. A wireline piston core barrel for sampling
cohesionless sand and gravel below the water table. Ground Water Mon-
itoring Review. Summer, Vol. 7, No. 3:74-82 (1987).
(15) Pankhurst, E.S. The isolation and enumeration of sulphate-reduc-
ing bacteria, p. 223-240. In: D.A. Shapton and R.G. Board (eds.), Isolation
of Anaerobes. Academic, NY (1971).
(16) Russell, D.J. and B. McDuffie. Analysis for phthalate esters in
environmental samples: Separation from PCBs and pesticides using dural
column chromatography. International Journal of Environmental Analyt-
ical Chemistry 15:165-183 (1983).
(17) Schellenberg, K. et al. Sorption of chlorinated phenols by natural
sediments and aquifer materials. Environmental Science and Technology.
18:652-657 (1984).
(18) Sims, R.C. Land treatment of polynuclear wastes. Ph.D. disserta-
tion. North Carolina State University, Raleigh, NC (1982).
(19) Stott, D.E. and M.A. Tabatabai. Identification of phospholipids
in soils and sewage sludges by high-performance liquid chromatography.
Journal of Environmental Quality. 14:107-110 (1985).
(20) United States Department of Agriculture. Soil survey laboratory
methods and procedures for collecting soil samples. Soil Survey Investiga-
tions Report No. 1. Soil Conservation Service. Soil Survey Investigation
(1972).
(21) United States Department of Agriculture. Soil taxonomy: a basic
system of soil classification for making and interpreting soil surveys. Agri-
cultural Handbook 436. Soil Conservation Service (1975).
-------
(22) U.S. Environmental Protection Agency (EPA). Test Methods for
Evaluating Solid Wastes: Physical/Chemical Methods. Second Edition.
SW-846. . EPA, Washington, DC (1982).
(23) Watwood, M.E. et al. Sulfur processing in forest soil and litter
along an elevational and vegetative gradient. Canadian Journal of Forest
Resources. 16:689-695 (1986).
(24) Wilson, J.T. et al. Influence of microbial adaptation of the fate
of organic pollutants in ground water. Environmental Toxicology and
Chemistry. 4:721-726 (1985).
(25) Wilson, B.H. et al. Biotransformation of selected alkylbenzenes
and halogenated aliphatic hydrocarbons in methanogenic aquifer material:
A microcosm study. Environmental Science and Technology. 20:997-1002
(1986).
(26) Wilson, J.T. et al. Enumeration and characterization of bacteria
indigenous to a shouldow water-table aquifer. Groundwater 21:134-142
(1983).
10
------- |