[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR796.3100]
[Page 92-96]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 796_CHEMICAL FATE TESTING GUIDELINES--Table of Contents
Subpart D_Transformation Processes
Sec. 796.3100 Aerobic aquatic biodegradation.
(a) Introduction--(1) Purpose. (i) This Guideline is designed to
develop data on the rate and extent of aerobic biodegradation that might
occur when chemical substances are released to aquatic environments. A
high biodegradability result in this test provides evidence that the
test substance will be biodegradable in natural aerobic freshwater
environments.
(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 test substance at the test concentration
may be observed. In such cases, further work is needed to assess the
aerobic aquatic 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) Definitions. (i) ``Adaptation'' is the process by which a
substance induces the synthesis of any degradative enzymes necessary to
catalyze the transformation of that substance.
(ii) ``Ultimate Biodegradability'' is the breakdown of an organic
compound to CO2, water, the oxides or mineral salts of other
elements and/or to products associated with normal metabolic processes
of microorganisms.
(iii) ``Ready Biodegradability'' is an expression used to describe
those substances which, in certain biodegradation test procedures,
produce positive results that are unequivocal and which lead to the
reasonable assumption that the substance will undergo rapid and ultimate
biodegradation in aerobic aquatic environments.
(3) Principle of the test method. This Guideline method is based on
the method described by William Gledhill (1975) under paragraph (d)(1)
of this section. The method consists of a 2-week inoculum buildup period
during which soil and sewage microorganisms are provided the opportunity
to adapt to the test compound. This inoculum is added to a specially
equipped Erlenmeyer flask containing a defined medium with test
substance. A reservoir holding barium hydroxide solution is suspended in
the test flask. After inoculation, the test flasks are sparged with
CO2-free air, sealed, and incubated, with shaking in the
dark. Periodically, samples of the test mixture containing water-soluble
test substances are analyzed for dissolved organic carbon (DOC) and the
Ba(OH)2 from the reservoirs is titrated to measure the amount
of CO2 evolved. Differences in the extent of DOC
disappearance and CO2 evolution between control flasks
containing no test substance, and flasks containing test substance are
used to estimate the degree of ultimate biodegradation.
(4) Prerequisites. The total organic carbon (TOC) content of the
test substance shall be calculated or, if this is not possible,
analyzed, to enable the percent of theoretical yield of carbon dioxide
and percent of DOC loss to be calculated.
(5) Guideline information. (i) Information on the relative
proportions of the major components of the test substance 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 concentrations.
(6) Reference substances. Where investigating a chemical substance,
reference compounds may be useful and an inventory of suitable reference
compounds needs to be identified. In order to check the activity of the
inoculum the use of a reference compound is desirable. Aniline, sodium
citrate, dextrose, phthalic acid and trimellitic acid will exhibit
ultimate biodegradation under the conditions of this Test Guideline
method. These reference substances must yield 60 percent of theoretical
maximum CO2 and show a removal of 70 percent DOC within 28
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days. Otherwise the test is regarded as invalid and shall be repeated
using an inoculum from a different source.
(7) Reproducibility. The reproducibility of the method has not yet
been determined; however it is believed to be appropriate for a
screening test which has solely an acceptance but no rejective function.
(8) Sensitivity. The sensitivity of the method is determined by the
ability to measure the endogenous CO2 production of the
inoculum in the blank flask and by the sensitivity limit of the
dissolved organic carbon analysis. If the test is adapted to handle
\14\C-labeled test substances, test substance concentrations can be much
lower.
(9) Possibility of standardization. This possibility exists. The
major difficulty is to standardize the inoculum in such a way that
interlaboratory reproducibility is ensured.
(10) Possibility of automation. None at present, although parts of
the analyses may be automated.
(b) Test procedures--(1) Preparations--(i) Apparatus. The shake
flask apparatus under the following Figure 1 contains 10 mL of 0.2N
Ba(OH)2 in an open container suspended over 1 liter of
culture medium in a 2-liter Erlenmeyer flask.
[GRAPHIC] [TIFF OMITTED] TC01AP92.039
Figure 1--Shake-Flask System for Carbon Dioxide Evolution
The Ba(OH)2 container is made by placing a constriction just
above the 10 mL mark of a 50 mL heavy-duty centrifuge tube and attaching
the centrifuge tube to a 2 mm I.D. x 9 mm O.D. glass tube by means of 3
glass support rods. The centrifuge tube opening is large enough to
permit CO2 to diffuse into the Ba(OH)2, while the
constriction permits transferal of the flask to and from the shaker
without Ba(OH)2 spillage into the medium. For periodic
removal and addition of base from the center well, a polypropylene
capillary tube, attached at one end to a 10 ml disposable syringe, is
inserted through the 9 mm O.D. glass tube into the Ba(OH)2
reservoir. The reservoir access port is easily sealed during incubation
with a serum bottle stopper. Two glass tubes are added for sparging,
venting, and medium sampling. The tops of these
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tubes are connected with a short section of flexible tubing during
incubation.
(ii) Reagents and stock solutions. (A) Stock solutions, I, II, and
III under the following Table 1.
(B) Yeast extract.
(C) Vitamin-free casamino acids.
(D) 70 percent O2 in nitrogen or CO2-free air.
(E) 0.2N Ba(OH)2.
(F) 0.1 N HCl.
(G) 20 percent H2SO4.
(H) Phenolphthalein.
(I) Dilution water--distilled, deionized water (DIW).
(iii) Soil inoculum. A fresh sample of an organically rich soil is
used as the inoculum in the ultimate biodegradation test. Soil is
collected, prepared, and stored according to the recommendations of
Pramer and Bartha (1972) under paragraph (d)(2) of this section. The
soil surface is cleared of litter and a soil sample is obtained 10 to 20
cm below the surface. The sample is screened through a sieve with 2 to 5
mm openings and stored in a polyethylene bag at 2 to 4 [deg]C for not
more than 30 days prior to use. The soil is never allowed to air-dry,
and shall not be frozen during storage.
Table 1--Medium Employed for Assay of CO2 Evolution
------------------------------------------------------------------------
Stock
Solution
Solution \1\ Compound Conc. (g/
L)
------------------------------------------------------------------------
I NH4Cl............................ 35
KNO3............................. 15
K2HPO4[middot]3H2O............... 750
NaH2PO4[middot]H2O............... 25
II \2\ KCl.............................. 10
MgSO4............................ 20
FeSO4[middot]7H2O................ 1
III CaCl2............................ 5
ZnCl2............................ 0.05
MnCl2[middot]4H2O................ 0.5
CuCl2............................ 0.05
CoCl2............................ 0.001
H3 BO3........................... 0.001
MoO3............................. 0.0004
------------------------------------------------------------------------
\1\= Each liter of test medium contains 1 mL of each solution.
\2\= Final pH is adjusted to 3.0 with 0.10 N HCl.
(iv) Acclimation Medium. Acclimation medium is prepared by adding,
for each liter of distilled, deionized water (DIW): 1 mL each of
solutions I, II, and III in Table 1 in paragraph (b)(1)(iii) of this
section, 1.0 gm of soil inoculum (prepared according to paragraph
(b)(1)(iii) of this section), 2.0 mL of aerated mixed liquor (obtained
from an activated sludge treatment plant not more than 2 days prior to
commencing the acclimation phase, and stored in the interim at 4 [deg]C)
and 50 mL raw domestic influent sewage. This medium is mixed for 15
minutes and filtered through a glass wool plug in a glass funnel. The
filtrate is permitted to stand for 1 hour, refiltered through glass
wool, and supplemented with 25 mg/L each of Difco vitamin-free casamino
acids and yeast extract. Appropriate volumes are added to 2-liter
Erlenmeyer flasks. Test compounds are added incrementally during the
acclimation period at concentrations equivalent to 4, 8, and 8 mg/L
carbon on days 0, 7, and 11, respectively. On day 14, the medium is
refiltered through glass wool prior to use in the test. For evaluating
the biodegradability of a series of functionally or structurally related
chemicals, media from all inoculum flasks may be combined before final
filtration.
(2) Procedures. (i) Inoculum (100 mL of acclimation medium) is added
to 900 mL DIW containing 1 mL each of solutions I, II, and III in Table
1 under paragraph (b)(1)(iii) of this section in a 2-liter Erlenmeyer
flask. Test compound equivalent to 10 mg/liter carbon is added to each
of the replicate flasks containing the test medium. Ten mL of 0.2 N Ba
(OH)2 are added to the suspended reservoir in each flask and
duplicate 10 mL samples of Ba(OH)2 are also saved as
titration blanks for analysis with test samples. Flasks are sparged with
CO2-free air (for volatile test materials, sparging is done
prior to addition of the chemical), sealed, and placed on a gyrotary
shaker (approximately 125 rpm) at 20 to 25 [deg]C in the dark. For each
set of experiments, each test, reference, inhibited, and control system
should be analyzed at time zero and at a minimum of four other times
from time zero through day 28. Sampling must be made with sufficient
frequency to allow for a smooth plot of biodegradation with time.
Sampling times should be varied by the investigator as deemed
appropriate to match the rate of degradation of the test substance.
Tests may be terminated when biodegradation reaches a plateau and is
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consistent (10 percent) over 3 consecutive days or
on day 28, whichever occurs first. For chemicals which are water soluble
at the test concentration, an adequate volume (5 to 10 mL) of medium is
removed for DOC analysis. Each sample for DOC analysis should be
filtered through a membrane filter of 0.45 micrometer pore diameter
before DOC analysis. For all test and reference compounds,
Ba(OH)2 from the center well is removed for analysis. The
center well is rinsed with 10 mL CO2-free DIW and is refilled
with fresh base. Rinse water is combined with the Ba(OH)2
sample to be analyzed. Flasks are resealed and placed on the shaker. On
the day prior to terminating the test, 3 mL of 20 percent
H2SO4 are added to the medium to release carbonate
bound CO2.
(ii) For each set of experiments, each test substance shall be
tested in triplicate.
(iii) For each set of experiments, one or two reference compounds
are included to assess the microbial activity of the test medium.
Duplicate reference flasks are prepared by adding reference compound
equivalent to 10 mg/liter carbon to each of two flasks containing the
test medium. Reference compounds which are positive for ultimate
biodegradability include: sodium citrate, dextrose, phthalic acid,
trimellitic acid, and aniline.
(iv) For each test set, triplicate controls receiving inoculated
medium and no test compound, plus all test and reference flasks, are
analyzed for CO2 evolution and DOC removal. Results from
analysis of the control flasks (DOC, CO2 evolution, etc.) are
subtracted from corresponding experimental flasks containing test
compound in order to arrive at the net effect due to the test compound.
(v) A test system containing a growth inhibitor should be
established as a control for each substance tested for biodegradation by
this method. That inhibited system must contain the same amount of
water, mineral nutrients, inoculum, and test substance used in the
uninhibited test systems, plus 50 mg/L mercuric chloride
(HgCl2) to inhibit microbial activity.
(vi) Flasks shall be incubated in the dark to minimize both
photochemical reactions and algal growth. Appropriate sterile controls
or controls containing a metabolic inhibitor, such as 50 mg/1
HgCl2, are needed to correct for interferences due to
nonbiological degradation. With volatile organic materials, sparging
with CO2-free air is performed only once, just prior to
addition of the test chemical. Analyses for CO2 evolution and
DOC removal are conducted within 2 to 3 hours of sampling to minimize
interferences which may occur in storage. All glassware should be free
of organic carbon contaminants.
(3) Analytical measurements. The quantity of CO2 evolved
is measured by titration of the entire Ba(OH)2 sample (10 mL
Ba(OH)2+10 mL rinse water) with 0.1 N HCl to the
phenolphthalein end point. Ba(OH)2 blanks are also
supplemented with 10 mL CO2-free DIW and titrated in a
similar manner. Samples (5 mL) for DOC are centrifuged and/or filtered
and supernatant or filtrate analyzed by a suitable total organic carbon
method.
(c) Data and reporting--(1) Treatment of results. (i) Test compound
(10 mg carbon) is theoretically converted to 0.833 mmol CO2.
Absorbed CO2 precipitates as BaCO3 from
Ba(OH)2, causing a reduction in alkalinity by the equivalent
of 16.67 mL of 0.1 N HCl for complete conversion of the test compound
carbon to CO2. Therefore, the percent theoretical
CO2 evolved from the test compound is calculated at any
sampling time from the formula:
Percent CO2 evolution=[(TF-CF)/16.67] 100 (for 10 mg/L test
compound carbon)
where:
TF= mL 0.1 N HCl required to titrate Ba(OH)2 samples from the
test flask
CF= mL 0.1 N HCl required to titrate Ba(OH)2 samples from the
control flask.
(ii) The cumulative percent CO2 evolution at any sample
time is calculated as the summation of the percent CO2
evolved at all sample points of the test.
(iii) The percent DOC disappearance from the test compound is
calculated from the following equation:
Percent DOC Removal=[1-(DTFx- DCFx)/
(DTFo- DCFo)] 100
where:
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DTF= Dissolved organic carbon from test flask
DCF= Dissolved organic carbon from control flask
o= Day zero measurements
x= Day of measurements during test.
(iv) The difference between the amount of 0.1 N HCl used for the
Ba(OH)2 titration blank samples and the Ba(OH)2
samples from the control units (no test compound) is an indication of
the activity of the microorganisms in the test system. In general, this
difference is approximately 1 to 3 mL of 0.1 N HCl at each sampling
time. A finding of no difference in the titration volumes between these
two samples indicates a poor inoculum. In this case, the validity of the
test results is questionable and the test set shall be rerun beginning
with the acclimation phase.
(v) CO2 evolution in the reference flasks is also
indicative of the activity of the microbial test system. The suggested
reference compounds should all yield final CO2 evolution
values of at least 60 percent of theoretical CO2. If, for any
test set, the percent theoretical CO2 evolution value for the
reference flasks is outside this range, the test results are considered
invalid and the test is rerun.
(vi) Inhibition by the test compound is indicated by lower
CO2 evolution in the test flasks than in the control flasks.
If inhibition is noted, the study for this compound is rerun beginning
with the acclimation phase. During the test phase for inhibitory
compounds, the test chemical is added incrementally according to the
schedule: Day 0--0.5 mg/liter as organic carbon, Day 2--1 mg/liter C,
Day 4--1.5 mg/liter C, Day 7--2 mg/liter C, Day 10--5 mg/liter C. For
this case, the Ba(OH)2 is sampled on Day 10, and weekly
thereafter. The total test duration remains 28 days.
(vii) The use of \14\C-labeled chemicals is not required. If
appropriately labeled test substance is readily available and if the
investigator chooses to use this procedure with labeled test substance,
this is an acceptable alternative. If this option is chosen, the
investigator may use lower test substance concentrations if those
concentrations are more representative of environmental levels.
(2) Test report. (i) For each test and reference compound, the
following data shall be reported.
(ii) Information on the inoculum, including source, collection date,
handling, storage and adaptation possibilities (i.e., that the inoculum
might have been exposed to the test substance either before or after
collection and prior to use in the test).
(iii) Results from each test, reference, inhibited (with
HgCl2) and control system at each sampling time, including an
average result for the triplicate test substance systems and the
standard deviation for that average.
(iv) Average cumulative percent theoretical CO2 evolution
over the test duration.
(v) Dissolved organic carbon due to test compound at each sampling
time (DTF-DCF).
(vi) Average percent DOC removal at each sampling time.
(vii) Twenty-eight day standard deviation for percent CO2
evolution and DOC removal.
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Gledhill, W.E. ``Screening Test for Assessment of Ultimate
Biodegradability: Linear Alkyl Benzene Sulfonate,'' Applied
Microbiology, 30:922-929 (1975).
(2) Pramer, D., Bartha, R. ``Preparation and Processing of Soil
Samples for Biodegradation Testing,'' Environmental Letters, 2:217-224
(1972).
[50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987]