United States       Prevention, Pesticides     EPA712-C-98-090
         Environmental Protection    and Toxic Substances     January 1998
         Agency        (7101)
&EPA    Fate, Transport and
          Transformation Test
          OPPTS 835.3400
          Biodegradability of
          Organic Chemicals

     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.).

     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 202-512-0132 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

OPPTS 835.3400  Anaerobic biodegradability of organic chemicals.
     (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  796.3140   Anaerobic
Biodegradability of Organic Chemicals.

     (b) Introduction—(1) Purpose, (i) This  guideline has been  devel-
oped for screening for anaerobic biodegradability of organic compounds.
A high biodegradability result in this test provides evidence that the test
substance will be  biodegradable in  sewage-treatment plant  anaerobic
digestors and in  many natural anaerobic  environments such as swamps,
flooded soils, and surface water sediments.

     (ii)  On the  contrary,  a low biodegradation result may have other
causes than poor biodegradability of the test substance. Inhibition  of the
microbial inoculum by the substance at the test concentration may be ob-
served.  In  such  cases further work is needed to  assess the  anaerobic
biodegradability and to determine the concentrations  at which toxic effects
are evident. An estimate of the expected environmental concentration will
help to put toxic effects into perspective.

     (2) Principle of the test method, (i) This portion of this guideline
is based on the biodegradability methods referenced under paragraph (e)
of this guideline.

     (ii) A  chemically defined anaerobic medium, containing resazurin as
an oxidation reduction indicator  and 10 percent (v/v) primary anaerobic
digestor sludge from a waste treatment plant, is dispensed in 100-mL por-
tions into  160-mL capacity  serum  bottles.  Selected bottles are supple-
mented with test substance  at a  concentration equivalent to 50 mg/L as
organic carbon.  Gas production is measured with a pressure transducer.
The  extent of biodegradation is  determined by comparing gas production
from blank control bottles and bottles containing the test substance.

     (iii) The average cumulative gas production (CH4 + CO2) is reported,
in milliliters, for blank controls, solvent controls, test substances, and any
reference compounds. Also reported is the percent of theoretical anaerobic
biodegradation at test completion or 56 days, whichever comes first, and
the standard deviation between replicate bottles.

     (3) Prerequisites. The total organic carbon content of the  test mate-
rial  should  be calculated or, if this is  not possible, analyzed, to enable
the theoretical yield of CH4 + CO2 to be calculated.

     (4) Guidance information, (i) Information on the relative proportions
of the major components of the test material will be useful in interpreting
the results obtained, particularly in those cases where the result lies close
to a "pass level".

     (ii) Information on the toxicity of the chemical may be useful in the
interpretation of low results and in the selection of appropriate test con-

     (5) Reference substances. In some cases, when investigating a sub-
stance, reference  substances may be useful and an inventory of suitable
reference substances needs to be identified. The use of a  reference sub-
stance is desirable in order to check the activity of the inoculum; ethanol
may be  used for this purpose. The ethanol must exhibit anaerobic biodeg-
radation (as gas production) greater than 50 percent of the theoretical max-
imum within 56 days,  otherwise the test is regarded as  invalid and must
be repeated using an inoculum from a different source.

     (6) Applicability. The method is only applicable to those organic test
substances which,  at the concentration used in the test,  are not inhibitory
to bacteria.

     (7) Reproducibility. The reproducibility of the method has not yet
been determined; however, it is believed to be appropriate for a screening

     (8) Sensitivity. The sensitivity  of the method is largely determined
by the necessity to compare gas production in test substance bottles with
gas production in  blank control bottles. The method suggests the use of
test substances at a concentration of 50 mg/L as organic carbon. This con-
centration will produce a maximum of  10.5 mL of CtL;  +  CCh at 35 °C.
Actual measured gas production will be less due to incomplete conversion
of all the organic  carbon into CfU and CCh and the extent to which the
CCh and CtL; remain  solubilized  in the aqueous phase. The use of test
substance at 50 mg/L as organic carbon represents a compromise between
the need to maximize  gas production and thus the sensitivity of the test,
and the  need to minimize the possibility of toxicity to the microbial popu-

     (c) Test procedures—(1) Preparations—(i)  Required apparatus.
(A) If gas production  is measured with a pressure  transducer, apparatus
such as  a 20-gauge syringe needle attached by means of an inert capillary
tube to a 3-way valve  (Hamilton Mininert valve 3-FLM-IX or equivalent)
fitted to a pressure transducer (Unimeasure 100-500Q/2mA pressure trans-
ducer or equivalent) and an  appropriate ohmmeter (e.g. Digitec Model

     (B) If gas production is measured with a syringe,  apparatus such as
a 20-mL capacity gas-tight glass syringe fitted with a  20-gauge syringe

     (C) If CH4 and CCh are quantified using an analytical procedure spe-
cific for these gases, apparatus needed to carry out that analysis, such as
a gas chromatograph fitted with a suitable detector.

     (D) An incubator  sufficient to store the test bottles at 35+ 1 °C for
the duration of the test.

     (E) Apparatus  suitable for the maintenance of anaerobic  conditions
during medium preparation and inoculation, such as that shown in the fol-
lowing Figure 1.

     (F) A supply of 160-mL capacity serum bottles with butyl rubber

     (ii) Nutrient medium—(A)  Stock solutions. (7) S-l: Prepare a solu-
tion in distilled water containing resazurin at 0.5 g/L.

     (2)  S-2:  Dissolve  20 g  ammonium monohydrogen  phosphate
(NH4)2HPO4 and 100 g of ammonium chloride, NH4C1, in distilled water
and dilute to 1 L.

     (3) S-3: Dissolve 18 g calcium chloride, CaCk, 180 g magnesium
chloride  hexahydrate,  MgCi2-6Fi2O,  130  g potassium  chloride, KC1,
2 g manganous chloride tetrahydrate, MnCl2-4H2O,  3 g cobaltous chloride
tetrahydrate, CoCi2 4H2O, 0.6 g boric acid, HsBOs,  0.23 g cupric chloride,
CuCi2, 1.0 g sodium molybdate dihydrate, Na2MoC>4 2H2O, and 0.2 g zinc
chloride, ZnCk, in distilled water and dilute to 1 L.

     (4) S-4: Dissolve 368  g ferrous  chloride tetrahydrate, FeCl2-4H2O,
in distilled water and dilute to 1 L.

     (5) S-5: Dissolve 50 g hydrated sodium sulfide, Na2S-92O in distilled
water and dilute to 1 L.

     (B) Reagents. Sodium bicarbonate, NaHCOs.

     (iii) Inoculum. (A) The inoculum should consist of sludge from an
anaerobic sludge digestor. It  is recommended that  well-mixed primary
sludge from a digestor with a sludge retention time  of  15 to 25 days be
used. At the time of collection the sludge should be  sieved through a
2-mm mesh screen.

     (B)  Most sludges can be stored for up to 1 to 2 weeks at 4  °C if
necessary, but it is recommended that fresh sludge be used.

     (C) Care should be taken to minimize exposure of the sludge to oxy-
gen during collection, handling, and storage.

     (2) Procedure—(i) Inoculated  medium. (A) Prereduced medium is
prepared by  adding  8  mL of stock solution S-l,  8 mL  of S-2, and
40  mL of S-3  to approximately  3,500 mL of deionized water in a
4-L Florence or Erlenmeyer flask.  This medium is heated to a boil,  while
being stirred with a magnetic stir bar and sparged with oxygen-free  nitro-
gen. Oxygen-free nitrogen is obtained by passing nitrogen gas through a
quartz cylinder filled with copper filings  heated to 600 °C. Alternatively,
commercial oxygen-free nitrogen may be used.

     (B)  The flask containing the  medium is placed in an  ice bath and
oxygen-free  CC>2 is introduced into the stream of oxygen-free nitrogen to
a concentration in the gas stream  of 30 percent (v/v).

     (C)  When the medium has cooled  to 35 °C, the  flask is removed
from the ice bath and the following components are added: 4 mL of solu-
tion S-4, 40  mL of solution  S-5, 10.56 g sodium  bicarbonate, and
400  mL  of sludge inoculum. The  final volume should be approximately

     (ii)  Filling test  bottles. (A)  Portions  of  the  inoculated  medium
(100-mL) are transferred anaerobically into serum bottles with a total ca-
pacity of 160 mL. Referring to  Figure 1 under paragraph (c)(l)(i)(E) of
this  guideline, Vi and ¥2 are  valves that are used to control the transfer
of medium to the serum bottles. Inoculated medium is drawn  into the pipet
by suction, the pipet is moved,  and the  tip inserted into a  serum bottle.
During these processes, the serum bottle and neck of the medium flask
are  continually sparged with the  oxygen-free mixture of nitrogen and car-
bon dioxide.

     (B) The medium in the pipet is discharged into the serum bottle.


     (C) A new butyl rubber serum-bottle stopper is inserted into the neck
of the bottle while the needle used to sparge the contents with the Ni/
CO2 mixture is removed.

     (iii) Test and reference chemicals. (A) Test and reference chemicals
are added to serum  bottles to yield a final concentration of 50 mg/L  as
organic carbon. These chemicals may be  added to  the bottles prior to the
addition  of inoculated medium  or following addition, depending on the
nature of the  test or reference substance and whether or not it must  be
added to the bottles dissolved in a volatile solvent.

     (B) Test or reference chemicals with sufficiently high water solubility
may be added to test bottles from a neutralized stock solution. The stock
solution should be prepared so that a minimal volume is needed to yield
50 mg/L as organic carbon in the medium.

     (C) Suitable liquid test or reference chemicals may be added directly
by injection from a calibrated syringe.

     (D)  Test  or  reference chemicals with relatively low water solubility
may be added  to test bottles by a direct  addition  of weighed  amounts  or
by using an organic  solvent to  dissolve  the  chemical  before  addition  to
test bottles. Direct addition is recommended.  If a  volatile organic solvent
is  used, a  suitable procedure is to dissolve the chemical in the solvent,
pipet an appropriate amount into the bottle, allow the solvent to evaporate,
and then add  the inoculated medium.  Diethyl ether is a suitable solvent
for many organics, but it must be completely removed from the bottle
because it  will adversely affect methanogenesis. If an organic solvent is
used without removal of the solvent before the test, the solvent must nei-
ther  significantly inhibit nor contribute to apparent gas production. Aceto-
nitrile, dioxane, and pyridine have been found acceptable  for this purpose.

     (E)  Bottles  containing inoculated  medium but no test or reference
chemical are employed in each test. These are the blank controls.

     (F) If an  organic solvent is used to add chemical to test bottles with-
out evaporation of the solvent before the test, bottles containing inoculated
medium and an equivalent amount of the organic  solvent, but no test  or
reference  chemical, must be employed for each solvent  used. These are
the solvent controls.

     (G) The substance bottles, blank controls, and  solvent controls should
be prepared in triplicate.

     (iv) Incubation. (A) At the start  of the  incubation,  pressure in each
bottle must be released.

     (B)  Bottles  are incubated in the  dark at 35+1 °C for 8 weeks  or
until biodegradation  is complete.  Bottles containing oxidized (pink) resaz-
urin should be discarded.

     (3) Analytical measurements, (i) A sufficient number of measure-
ments of gas pressure or gas volume should be made to establish the pat-
tern of gas production with time. Measurements are generally made weekly
for up to 8 weeks. The frequency of measurements may be varied by the
investigator  as deemed  appropriate  to match the degradation rate of the

     (ii) Gas production is measured for each bottle using a pressure trans-
ducer, syringe, or other suitable apparatus.

     (iii) The use of a pressure transducer is recommended. The ohmmeter
should be calibrated by injecting known volumes of gas into bottles con-
taining medium  and a standard  curve  of gas volume  vs. meter  reading
plotted.  Excess pressure should be vented after each measurement so that
all bottles will have  the same internal  pressure following each measure-
ment time.

     (iv) If a syringe is used to measure gas volume, the following proce-
dure is recommended.  The syringe  is flushed with 30  percent (v/v) CO2
in oxygen-free nitrogen. The syringe is held in a horizontal position during
the measurement, taking care to  keep the needle within the gas space of
the serum bottle. Gas production is determined by allowing  the  syringe
plunger to move freely to equalize  the  vessel and atmospheric pressure.

     (v) CH4 and CO2 may be determined using analytical methods suit-
able  for the detection and quantification  of those compounds.

     (d) Data and reporting—(1) Treatment of results, (i)  Cumulative
average gas volume from the anaerobic  biodegradation of test or reference
substances is calculated by subtracting the cumulative average  gas  volume
production for triplicate blank controls  (or solvent controls, if an  organic
solvent was included) from the average value for triplicate test or reference
substance  bottles at the same incubation time. The percent of theoretical
gas volume produced is calculated by dividing cumulative average gas vol-
ume  from test or reference chemical by the theoretical maximum gas pro-
duction and multiplying by 100.

     (ii) The maximum CH4 +  CO2 production theoretically obtainable
from an organic  chemical in this  test is  10.5 mL, if the  starting concentra-
tion is 50 mg/L as organic carbon. This  can be calculated as shown below,
using benzoic acid (CyHeCh) as an example.
C7H602 + 12 H20	> 7 C02 + 15 H2

7 CO2 + 15 H2	> 3.75 CH4 + 3.25 CO2 + 7.5 H2O

C7H6O2 + 4.5 H2O	> 3.75 CH4 + 3.25 CO2


At a concentration of 50 mg/L  as  organic carbon in a 100 mL aqueous
phase at 35 °C, the maximum volume of gas produced is calculated as

At 50  mg/L as organic  carbon in  a 100 mL there is 7.27 mg benzoic

    7.27 mg benzoic acid = 0.0595  mmol

    0.0595 mmol x 3.75 = 0.2232 mmol CH4

    0.0595 x 3.25 = 0.1934 mmol CO2

    0.2232 + 0.1934 = 0.4167 mmol total gas production.

At 35  °C and  atmospheric  pressure,  one mole of gas occupies  approxi-
mately 25.25 L, thus 0.4167 mmol will occupy 10.5 mL.

    (iv) Therefore, any test compound  added at a concentration that pro-
vides  5 mg of organic  carbon to the test bottle will have a theoretical
maximum gas production of 10.5 mL.

    (2) Test report. The following  must be reported:

    (i) Information on the inoculum including information on the source,
retention time,  percent volatile solids, date of collection, storage, handling
and adaptation possibilities (i.e.  information  on the possibility that the
inoculum  was  exposed  to the test  chemical or related chemicals before
the test). Retention time and percent volatile solids of the sludge  can usu-
ally be obtained from the treatment plant operator.

    (ii) Average cumulative gas production in milliliters from blank con-
trol bottles, solvent control bottles, test substance bottles, and  reference
compound bottles at each measurement time.

    (iii) Percent of theoretical anaerobic bio degradation for each test sub-
stance and reference compound at each measurement time.

    (iv) The standard deviation for  each replicate  set of bottles at the
final measurement time.

    (v) A plot of the percent of theoretical anaerobic biodegradation vs.
time for each test substance and reference compound.

    (vi) A  description of any deviation from this test guideline, such as
variations in the medium or the concentration of test substance, test condi-
tions, or analytical techniques.

    (e) References. The following  references should be consulted for ad-
ditional background material on this test guideline.

     (1) Environmental Protection Agency. J.B.  Healy and L.Y. Young,
Methanogenic biodegradation of aromatic compounds. Workshop on mi-
crobial degradation of pollutants in marine environments. EPA Report 6007
9-79-012. Gulf Breeze, Florida (1978).

     (2) Gossett, J.M. et al. Heat treatment of refuse for increasing anaer-
obic biodegradability.  Stanford  University Civil Engineering Technical
Report No. 205(1976).

     (3) Healy, J.B.  et al. Heat treatment of organics for increasing anaer-
obic biodegradability.  Stanford  University Civil Engineering Technical
Report No. 222(1977).

     (4) Healy, J.B.  and L.Y. Young. Degradation of simple aromatic com-
pounds  under methanogenic conditions. Abstracts of the Annual Meeting
of the American Society for Microbiology.  13:263  (1977).

     (5) Owen, W.F. et al. Bioassay for monitoring biochemical methane
potential and anaerobic toxicity.  Water Research 13:485-492 (1979).

     (6) Healy, J.B. and L.Y. Young. Anaerobic biodegradation  of eleven
aromatic compounds to methane. Applied and Environmental Microbiology
38:84-89 (1979).

     (7) Miller, T.C. and M.J. Wolin. A serum bottle modification of the
Hungate technique  for cultivating obligate anaerobes. Applied Microbi-
ology 27:985-987 (1974).