United States Prevention, Pesticides EPA712-C-98-076
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport, and
Transformation Test
Guidelines
OPPTS 835.3110
Ready Biodegradability
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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 835.3110 Ready biodegradability.
(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 40 CFR 796.3180 Ready
Biodegradability: Modified AFNOR Test, 796.3200 Ready
Biodegradability: Closed Bottle Test, 796.3220 Ready Biodegradability:
Modified MITI Test (I), 796.3240 Ready Biodegradability: Modified
OECD Test, and 796.3260 Ready Biodegradability: Modified Sturm Test,
and OECD 301A DOC Die-Away Test, 301 B CO2 Evolution Test, 301C
Modified MITI Test (I), 301 D Closed Bottle Test, 301 E Modified OECD
Screening Test, and 301 F Manometric Respirometry Test.
(b) Preliminary information. (1) In this guideline six methods are
described that permit the screening of chemicals for ready biodegradability
in an aerobic aqueous medium. They are:
(i) DOC Die-Away under paragraph (1) of this guideline.
(ii) CO2 Evolution (Modified Sturm Test) under paragraph (m) of
this guideline.
(iii) MITI (I) (Ministry of International Trade and Industry, Japan)
under paragraph (n) of this guideline.
(iv) Closed Bottle under paragraph (o) of this guideline.
(v) Modified OECD Screening under paragraph (p) of this guideline.
(vi) Manometric Respirometry under paragraph (q) of this guideline.
(2) The DOC die-away method is similar to the ISO Standard 7827-
1984 and replaces the Modified AFNOR method. The methods under para-
graphs (m), (n), (o), and (p) of this guideline are modified versions of
earlier OECD guidelines adopted in 1981. The manometric respirometry
method under paragraph (q) of this guideline is similar to the MITI (I)
method, differing mainly in the inocula employed.
(3) Much experience has accumulated with the six methods over the
years including an OECD interlaboratory comparison exercise (ring test)
in 1988. The accumulated experience, and the ring test, have confirmed
that the methods may be used for the assessment of ready biodegradability.
However, depending on the physical characteristics of the substance to
be tested, a particular method may be preferred.
(4) General considerations including those common to all six methods
are given under paragraphs (c) through (k) of this guideline. Details of
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individual methods are given under paragraphs (1) through (q) of this
guideline.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions and abbreviations also apply to this test guideline.
Biochemical oxygen demand (BOD) is the amount of oxygen in milli-
grams consumed by microorganisms when metabolizing a test compound;
also expressed as milligrams of oxygen uptake per milligram of test
compound.
Chemical oxygen demand (COD) is the amount of oxygen in milli-
grams consumed during oxidation of a test compound with hot, acidic di-
chromate; it provides a measure of the amount of oxidizable matter
present; also expressed as milligrams of oxygen consumed per milligram
of test compound.
Degradation phase is the time from the end of the lag period to the
time when 90 percent of the maximum level of degradation has been
reached.
Dissolved organic carbon (DOC) is the organic carbon present in so-
lution or that which passes through a 0.45 (im filter or remains in the
supernatant after centrifuging at approximately 4,000 xg (about
40.000 m sec-2) for 15 min.
Dissolved oxygen (DO) is the concentration of oxygen dissolved in
an aqueous sample expressed as milligrams per liter.
EC50 is the concentration of the test substance at which the respira-
tion rate is 50 percent of that shown by the control under the conditions
of the guideline for the activated sludge respiration inhibition test (OPPTS
850.6800; ISO Standard 8192).
Inherently biodegradable is a classification of chemicals for which
there is unequivocal evidence of biodegradation (primary or ultimate) in
any test of biodegradability.
Inorganic carbon is abbreviated 1C.
Lag phase is the period from inoculation in a die-away test until the
degradation percentage has increased to about 10 percent. The lag time
is often variable and poorly reproducible.
Organic carbon is abbreviated OC.
Primary biodegradation. is the alteration in the chemical structure
of a substance, brought about by biological action, resulting in the loss
of a specific property of that substance.
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Readily biodegradable is an arbitrary classification of chemicals
which have passed certain specified screening tests for ultimate
biodegradability; these tests are so stringent that it is assumed that such
compounds will rapidly and completely biodegrade in aquatic environ-
ments under aerobic conditions.
Suspended solids is abbreviated SS.
10-day window is the 10 days immediately following the attainment
of 10 percent biodegradation.
Test substance is abbreviated TS.
Theoretical carbon dioxide (ThCO^) is the quantity of carbon dioxide
in milligrams calculated to be produced from the known or measured car-
bon content of the test compound when fully mineralized; also expressed
as milligrams of carbon dioxide evolved per milligram of test compound.
Theoretical oxygen demand (ThOD) is the total amount of oxygen
in milligrams required to oxidize a chemical completely; it is calculated
from the molecular formula presented under paragraph (k)(2)(i) of this
guideline and is also expressed as milligrams of oxygen required per milli-
gram of test compound.
Total carbon (TC) is the sum of the organic carbon and the inorganic
carbon present in a sample.
Total organic carbon (TOC) of a sample is the sum of the organic
carbon in solution and in suspension.
Treatability is the amenability of compounds to removal during bio-
logical wastewater treatment without adversely affecting the normal oper-
ation of the treatment processes. Generally, readily biodegradable com-
pounds are treatable, but this is not the case for all inherently biodegrad-
able compounds. Abiotic processes may also operate.
Ultimate biodegradation (aerobic) is the level of degradation
achieved when the test compound is totally utilized by microorganisms
resulting in the production of carbon dioxide, water, mineral salts, and
new microbial cellular constituents (biomass).
(d) General principle of the tests. (1) A solution, or suspension,
of the test substance in a mineral nutrient medium is inoculated and incu-
bated under aerobic conditions in the dark or in diffuse light. The amount
of DOC in the test solution due to the inoculum should be kept as low
as possible compared with the amount of OC due to the test substance.
Allowance is made for the endogenous activity of the inoculum by running
parallel blanks with inoculum but without test substance, although the en-
dogenous activity of cells in the presence of a chemical will not exactly
match that in the endogenous control. A reference compound is run in
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parallel to check the activity of the inoculum and to serve as a general
check on the operation of the procedures.
(2) In general, degradation is followed by the determination of param-
eters such as DOC, CCh production and oxygen uptake, and measurements
are taken at sufficiently frequent intervals to allow the identification of
the beginning and end of biodegradation. With automatic respirometers
the measurement is continuous. DOC is sometimes measured in addition
to another parameter but this is usually done only at the beginning and
end of the test. Specific chemical analysis can also be used to assess pri-
mary degradation of the test substance and to determine the concentration
of any intermediate substances formed. It is obligatory in the MITI method
under paragraph (n) of this guideline.
(3) Normally, the test lasts for 28 days. However, tests may be ended
before 28 days, i.e. as soon as the biodegradation curve has reached a
plateau for at least three determinations. Tests may also be prolonged be-
yond 28 days when the curve shows that biodegradation has started but
that the plateau has not been reached by day 28, but in such cases the
chemical would not be classified as readily biodegradable.
(e) Information on the test substance. (1) In order to select the most
appropriate method, information on the chemical's solubility, vapor pres-
sure and adsorption characteristics is essential. The chemical structure or
formula should be known in order to calculate theoretical values and/or
check measured values of parameters, e.g. ThOD, ThCO2, DOC, TOC,
and COD. Information on the purity or the relative proportions of major
components of the test material is required in order to interpret the results
obtained, especially when the result lies close to the pass level.
(2) Information on the toxicity of the test substance to bacteria may
be very useful for selecting appropriate test concentrations and may be
essential for the correct interpretation of low biodegradation values. When
a chemical is subjected to ready biodegradability testing and appears to
be nonbiodegradable, the following procedure is recommended if a distinc-
tion between inhibition and inertness is desired (refer to paragraph (r)(l)
of this guideline).
(i) Similar or identical inocula should be used for the toxicity and
biodegradation tests.
(ii) To assess the toxicity of chemicals studied in ready
biodegradability tests, the application of one or a combination of the inhi-
bition of sludge respiration rate (OPPTS 850.6800; ISO Standard 8192),
BOD and/or growth inhibition methods is appropriate.
(iii) If inhibition due to toxicity is to be avoided, it is suggested that
the test substance concentrations used in ready biodegradability testing
should be less than 0.1 of the EC50 values (or less than EC20 values)
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obtained in toxicity testing. Compounds with an EC50 value greater than
300 mg/L are not likely to have toxic effects in ready biodegradability
testing.
(iv) EC50 values of less than 20 mg/L are likely to pose serious prob-
lems for the subsequent testing. Low test concentrations should be em-
ployed, necessitating the use of the stringent and sensitive Closed Bottle
test (under paragraph (o) of this guideline) or the use of 14C-labeled mate-
rial. Alternatively, an inoculum previously exposed to the test substance
may permit higher test substance concentrations to be used. In the latter
case, however, the stringency criterion of the ready biodegradability test
is lost.
(f) Applicability and selection of methods—(1) Evaluation of
biodegradability of test substances. Test substances that are soluble in
water to at least 100 mg/L and are nonvolatile and nonadsorbing may be
assessed by all methods described in this guideline. For those chemicals
that are poorly soluble in water, volatile or adsorbing, suitable methods
are indicated in Table 1. The manner in which poorly water-soluble chemi-
cals and volatile chemicals can be dealt with is described under paragraph
(f)(2) of this guideline, but in the MITI method under paragraph (n) of
this guideline neither solvents nor emulsifying agents can be used. Mod-
erately volatile chemicals may be tested by the DOC die-away method
under paragraph (1) of this guideline if there is sufficient gas space in
the test vessels (which should be suitably stoppered). In this case, an abi-
otic control must be set up to allow for any physical loss.
Table 1.—Applicability of Test Methods
Test
DOC Die-Away
CO2 Evolution
MITI (I)
Closed Bottle
Modified OECD Screening ..
Manometric Respirometry ...
Analytical method
Dissolved organic carbon
Respirometry: CO2 evolution
Respirometry: Oxygen con-
sumption
Respirometry: Dissolved ox-
ygen
Dissolved organic carbon
Oxygen consumption
Suitability for compounds that are:
poorly solu-
ble
+
+
+/-
+
volatile
+/-
+
+/-
adsorbing
+/-
+
+
+
+/-
+
(2) Evaluation of the biodegradability of poorly soluble com-
pounds. In biodegradability tests with poorly soluble compounds the fol-
lowing aspects should receive special attention. Additional information is
available in paragraphs (r)(2), (r)(3), (r)(4), and (r)(5) of this guideline.
(i) While homogeneous liquids will seldom present sampling prob-
lems, it is recommended that solid materials be homogenized by appro-
priate means to avoid errors due to nonhomogeneity. Special care must
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be taken when representative samples of a few milligrams are required
from mixtures of chemicals or substances with large amounts of impurities.
(ii) Various forms of agitation may be used during the test. Care
should be taken to use only sufficient agitation to keep the chemical dis-
persed, and to avoid overheating, excessive foaming, and excessive shear
forces.
(iii) An emulsifier which gives a stable dispersion of the chemical
may be used. It should not be toxic to bacteria and must not be bio-
degraded or cause foaming under test conditions.
(iv) The same criteria apply to solvents as to the emulsifiers.
(v) It is not recommended that solid carriers be used for solid test
substances, but they may be suitable for oily substances.
(vi) When auxiliary substances such as emulsifiers, solvents, and car-
riers are used, a blank run containing the auxiliary substance(s) should
be performed.
(vii) Any of the four respirometric tests (under paragraphs (m), (n),
(o), or (q) of this guideline) can be used to study the biodegradability
of poorly soluble compounds.
(g) Pass levels. The pass levels for ready biodegradability are 70 per-
cent removal of DOC and 60 percent of ThOD or ThCCh production for
respirometric methods. The pass levels are lower in the respirometric
methods because some of the carbon from the test chemical is incorporated
into new cells, and as a result, the percentage of CCh produced is lower
than the percentage of carbon being used. These pass values have to be
reached in a 10-day window within the 28-day period of the test, except
where mentioned below. The 10-day window begins when the degree of
biodegradation has reached 10 percent DOC disappearance, ThOD, or
ThCO2, and must end before day 28 of the test. Chemicals that reach
a pass level after the 28-day period are not deemed to be readily bio-
degradable. The 10-day window concept does not apply to the MITI meth-
od under paragraph (n) of this guideline. A pass criterion based on a
14-day window may be acceptable in the Closed Bottle method if it is
considered that the number of bottles necessary to evaluate the 10-day
window causes the test to become too unwieldy.
(h) Reference compounds. In order to check the activity of the
inoculum, one or more reference compounds that meet the criteria for
ready biodegradability are tested by setting up appropriate vessels in par-
allel as part of normal test runs. Suitable compounds are aniline (freshly
distilled), and phthalic or trimellitic acid, but sodium acetate, dextrose,
and sodium benzoate are probably too biodegradable to be useful for this
purpose.
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(i) Reproducibility of tests. Because of the nature of biodegradation
and of the mixed bacterial populations used as inocula, determinations
should be carried out at least in duplicate. It is usually found that the
larger the concentration of microorganisms initially added to the test me-
dium, the smaller will be the variation between replicates. Ring tests have
also shown that there can be large variations between results obtained by
different laboratories, but good agreement is normally obtained with easily
biodegradable compounds.
(j) General procedures and preparations—(1) General conditions.
General conditions applying to the methods are summarized in the follow-
ing Table 2. Apparatus and other experimental conditions pertaining spe-
cifically to an individual method are described later under the heading
for that method.
Table 2.—Test Conditions
Test
Concentration of TS
mg/L
mg DOC/L
mg ThOD/L
Concentration of
inoculum
mg/L SS
ml effluent/L
approx. cells/L
Concentration of ele-
ments in mineral
nutrient medium in
mg/mL
p
N
Na
K
Mq
Ca
Fe
PH
Temperature, °C
DOC
die-
away
10-40
<30
<100
1 07-1 08
116
1.3
86
122
22
9.9
0.05-0.1
7.4 + 0.2
22 + 2
C02
evo-
lution
10-20
<30
<100
1 07-1 08
116
1.3
86
122
22
9.9
0.05-0.1
7.4 + 0.2
22 + 2
MITI (I)
100
_
30
1 07-1 08
29
1.3
17.2
365
66
29.7
0.15
preferably 7
25 + 1
Closed
bottle
2-10
5-10
<5
1 Q4-1 06
11 6
0.13
8.6
122
22
9.9
0.05-0.1
7.4 + 0.2
22 + 2
Modified
OECD
screen-
ing
10-40
0.5
105
116
1.3
86
122
22
9.9
0.05-0.1
7.4 + 0.2
22 + 2
Manometric
respirometry
100
50-100
<30
<100
1 07-1 08
116
1.3
86
122
22
9.9
0.05-0.1
7.4 + 0.2
22 + 2
(2) Water. Deionized or distilled water, free from inhibitory con-
centrations of toxic substances (e.g. Cu2^ ions), is used. It must contain
no more than 10 percent of the OC content introduced by the test material.
The high purity of the test water is necessary in order to eliminate high
blank values. Contamination may result from inherent impurities and also
from the ion-exchange resins and lysed material from bacteria and algae.
For each series of tests, use only one batch of water, previously checked
by DOC analysis. Such a check is not necessary for the closed bottle meth-
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od, but the oxygen consumption of the water must be low (see paragraph
(o)(5)(ii) of this guideline).
(3) Mineral nutrient media. Mineral nutrient media are prepared
from stock solutions of appropriate concentrations of mineral components,
namely, potassium and sodium phosphates plus ammonium chloride, cal-
cium chloride, magnesium sulfate and iron(III) chloride. Since only a very
small inoculum, containing low concentrations of trace elements and
growth factors, is used in the Modified OECD Screening method under
paragraph (p) of this guideline, the medium for this test may need to be
fortified with additional nutrients. The details of the stock solutions of
mineral salts, trace elements and growth factors and the proportions used
are given under the headings for the separate tests.
(4) Methods of adding the test and reference substances. The
method used for adding the test and reference substances to the reaction
mixture depends upon the nature of the chemical, especially its water solu-
bility. For substances of adequate solubility (greater than about 1 g/L),
prepare stock solutions at appropriate concentrations and use aliquots to
prepare the final test solution. Dissolve less soluble substances in the min-
eral nutrient medium to avoid diluting the medium. Add substances that
are even less soluble directly to the final mineral nutrient medium. Finally,
refer to paragraph (f)(2) of this guideline for the handling of poorly soluble
substances, but note that in the MITI method under paragraph (n) of this
guideline neither organic solvents nor emulsifying agents are to be used.
(5) Inoculum. The inoculum may be derived from a variety of
sources: Activated sludge; sewage effluents (unchlorinated); surface waters
and soils; or a mixture of these. If activated sludge is used for the DOC
Die-Away, CCh Evolution, or Manometric Respirometry method, under
paragraphs (1), (m), and (q) of this guideline, it should be taken from a
treatment plant or laboratory-scale unit receiving predominantly domestic
sewage. Inocula from other sources, usually yielding lower cell densities,
have been found to give higher scattering of results. For the Closed Bottle
and Modified OECD Screening methods under paragraphs (o) and (p) of
this guideline, a more dilute inoculum without sludge floes is needed and
the preferred source is a secondary effluent from a domestic wastewater
treatment plant or laboratory-scale unit. For the MITI (I) method under
paragraph (n) of this guideline, the inoculum is derived from a mixture
of sources. Details of the sources and preparation of inocula are described
under the headings of the specific test methods.
(6) Preconditioning of inoculum. Inoculum may be preconditioned
to the experimental conditions, but not preadapted to the test substance.
Preconditioning consists of aerating activated sludge (in mineral nutrient
medium) or secondary effluent for 5-7 days at the test temperature. Pre-
conditioning sometimes improves the precision of the test methods by re-
8
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ducing blank values. It is considered unnecessary to precondition MITI
(I) inoculum under paragraph (n) of this guideline.
(7) Abiotic controls. When required, check for the possible abiotic
degradation of the test substance by determining the removal of DOC,
oxygen uptake or CCh evolution in sterile controls containing no inoculum.
Sterilize by filtration through a membrane (0.2-0.45 (im pore size) or by
the addition of a suitable toxic substance at an appropriate concentration.
If membrane filtration is used, collect samples aseptically to maintain ste-
rility. Unless adsorption of the test substance has been ruled out before-
hand, tests that measure biodegradation as the removal of DOC, especially
with activated sludge inocula, should include an abiotic control that is in-
oculated and poisoned.
(8) Number of flasks and samples, (i) At least two flasks or vessels
containing the test substance plus inoculum, and at least two containing
inoculum only should be used. Single vessels suffice for reference
compound plus inoculum and, when required, for toxicity, abiotic removal
and adsorption controls. The MITI (I) and Closed Bottle methods under
paragraph (n) and (o) of this guideline have special requirements for the
number of flasks. These are given under the specific headings for these
methods. It is mandatory to follow DOC and/or the other parameters in
the test suspension and inoculum blanks in parallel. It is advisable to fol-
low DOC in the other flasks in parallel as well. This may, however, not
always be possible.
(ii) Although it is necessary to ensure that sufficient samples or read-
ings are taken to allow the percentage removal in the 10-day window
to be assessed, it is not possible to specify accurately the frequency of
sampling because of the wide range of lag phases and rates of degradation
that may be observed. In the MITI method under paragraph (n) of this
guideline and the Manometric Respirometry method under paragraph (q)
of this guideline, if an automatic respirometer is used, sampling for oxygen
uptake presents no problems. In the latter method, daily readings are ade-
quate when nonautomatic respirometers are employed. Specific advice on
sampling is given under the headings of the other four tests.
(k) Data and reporting—(1) Treatment of results, (i) In the calcula-
tion of Dt (percent degradation), the mean values of the duplicate measure-
ment of the parameter in both test vessels and inoculum blank are used.
The formulas are set out under paragraphs (1) through (q) of this guideline.
The course of degradation is displayed graphically and the 10-day window
is indicated where applicable. Calculate and report the percent removal
achieved and the value at the plateau, or at the end of the test, and/or
at the end of the 10-day window, whichever is appropriate. In respiro-
metric methods, nitrogen-containing chemicals may affect oxygen uptake
because of nitrification (see paragraphs (k)(2) and (3) of this guideline).
If the ThOD cannot be calculated because the test material is insufficiently
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defined, the COD value may be used to calculate percent degradation.
However, it must be borne in mind that the COD is often not as high
as the ThOD as some chemicals are very poorly oxidized in the COD
test, resulting in falsely high values for percent biodegradation.
(ii) When specific chemical analytical data are available, calculate
primary biodegradation from:
Dt = (Sb - Sa)/Sb x 100
where:
Dt = percent primary degradation at time t, normally 28 days
Sa = residual amount of test chemical in inoculated medium at end
of the test (in milligrams or mg/L)
Sb = residual amount of test chemical in the abiotic control at the
end of the test (in milligrams or mg/L)
(2) Calculation and determination of suitable measures of ulti-
mate degradation. Depending on the method chosen, certain parameters
will be required. The following section describes the derivation of these
values. Additional information is available in paragraphs (r)(6), (r)(7), and
(r)(8) of this guideline. The use of these parameters is described in the
individual methods in paragraphs (1) through (q) of this guideline.
(i) Carbon content. The carbon content is calculated from the known
elemental composition or determined by elemental analysis of the test sub-
stance.
(ii) ThOD. The ThOD may be calculated if the elemental composition
is determined or known. For the compound
CcHhClclNnNanaOoPpSs
the ThOD, without nitrification, is
(16[2c + (h - cl - 3n)/2 + 3s + 5p/2 + na/2 - o] mg/mg)
molecular weight
With nitrification, the formula is
(16 [2c + (h - cl)/2 + 5n/2 + 3s + 5p/2 + na/2 - o]mg/mg)
ThOD
'N03
molecular weight
(iii) COD. The COD of water-soluble organic substances is deter-
mined by established procedures, e.g. according to ISO method 6060. COD
is often, and especially in the case of poorly soluble substances, deter-
10
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mined advantageously in a variant of the above analysis; i.e. in a closed
system with a pressure equalizer (refer to paragraph (r)(9) of this guide-
line). In this modification, compounds which are determined by the con-
ventional method (e.g. acetic acid) only with difficulty may often be suc-
cessfully quantified. This method also fails, however, in the case of pyri-
dine. If the potassium dichromate concentration is raised from 0.016 N
(0.0026 M) as prescribed by under paragraph (r)(9) of this guideline to
0.25 N (0.0416 M), the direct weighing-in of 5-10 mg of substance, which
is essential for the COD determination of poorly water-soluble substances,
is facilitated (see paragraph (r)(5) of this guideline).
(iv) DOC. DOC is, by definition, the organic carbon of any chemical
or mixture in water passing through a 0.45 (im pore size filter. Samples
from the test vessels are withdrawn and filtered immediately in the filtra-
tion apparatus using an appropriate membrane filter. The first 20 mL
(amount can be reduced when using small filters) of the filtrate are dis-
carded. Volumes of 10-20 mL or lower, if injected (volume depending
on the amount required for carbon analyzer) are retained for carbon analy-
sis. The DOC concentration is determined by means of an organic carbon
analyzer that is capable of accurately measuring a carbon concentration
equivalent to or lower than 10 percent of the initial DOC concentration
used in the test. Filtered samples that cannot be analyzed on the same
working day can be preserved by storage in a refrigerator at 4 °C. Pre-
served samples can be retained for 48 h before analysis, or for longer
at-18°C.
(v) Remarks. (A) Membrane filters are often impregnated with
surfactants for hydrophilization. Thus the filter may contain up to several
milligrams of soluble organic carbon which may interfere in the
biodegradability determinations. Surfactants and other soluble organic
compounds may be removed by boiling the filters in deionized water for
three periods each of 1 h unless this destroys the filters; washing is rec-
ommended in this case. The filters may then be stored in water for 1 week.
If disposable filter cartridges are used, each lot must be checked to confirm
that it does not release soluble organic carbon.
(B) Depending on the type of membrane filter, the test chemical may
be retained by adsorption. It may therefore be advisable to ensure that
the test chemical is not retained by the filter.
(C) Centrifugation at 4,000 x g (about 40.000 m sec 2) for 15 mm
may be used for differentiation of TOC versus DOC instead of filtration.
The method is not reliable at initial concentrations of <10 mg DOC/L
since either not all bacteria are removed or carbon as part of the bacterial
plasma is redissolved.
(3) Correction for interference by nitrification in determination
of biodegradation by oxygen uptake. Respirometric methods with oxy-
11
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gen uptake as the analytical procedure may be influenced significantly by
the oxygen uptake resulting from ammonium oxidation.
(i) Errors due to not considering nitrification in the assessment by
oxygen uptake of the biodegradability of test substances not containing
nitrogen are marginal (not greater than 5 percent), even if oxidation of
the ammonium ion nitrogen in the medium occurs erratically in test and
blank vessels. However, for test substances containing nitrogen, serious
errors can arise if the observed oxygen uptake is not corrected for the
amount of oxygen used in oxidizing ammonium ion to nitrite ion and ni-
trate ion. In the case of complete nitrification, or transformation of ammo-
nium ion to nitrate ion, the following equation applies:
NH4^ + 2 O2 > NO3 + 2HH + H2O
(ii) The oxygen taken up by 14 g of nitrogen is 64 g and thus the
oxygen consumed in nitrate formation is 4.57 x increase of nitrate ion
nitrogen concentration. If incomplete nitrification takes place, the follow-
ing equations apply:
NH4^ + 3/2 O2 > NO2 + 2HH + H2O
NO2 + 1/2 O2 > NO3
(iii) The oxygen taken up by 14 g of nitrogen in being oxidized to
nitrite is 48 g, i.e. a factor of 3.43x increase of nitrite ion nitrogen con-
centration.
(iv) Since the reactions are sequential, being carried out by distinct
and different bacterial species, it is possible for the concentration of nitrite
ion to increase or decrease during the test. In the latter case an equivalent
concentration of nitrate ion would be formed. Thus, the oxygen consumed
in the formation of nitrate ion is 4.57x the increase in concentration of
nitrate ion nitrogen, whereas the oxygen associated with the formation of
nitrite ion is 3.43x the increase in the concentration of nitrite ion nitrogen;
or, with the decrease in its concentration, the oxygen "loss" is 3.43 multi-
plied by the decrease in nitrite concentration.
(v) Alternatively, if only "total oxidized N" is determined, the oxy-
gen uptake due to nitrification may be taken to be, as a first approximation,
4.57 x increase in oxidized nitrogen.
(vi) The corrected value for oxygen consumption due to carbon oxida-
tion is then compared with ThODNH3, as calculated under paragraph
(k)(2)(ii) of this guideline.
(4) Validity of tests, (i) A test is considered valid if the difference
in extremes of replicate values of biodegradation of the test chemical at
the plateau, at the end of the test or at the end of the 10-day window,
as appropriate, is less than 20 percent, and if the percent degradation of
12
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the reference compound has reached the pass levels by day 14. If either
of these conditions is not met, the test should be repeated. Because of
the stringency of the methods, low values do not necessarily mean that
the test substance is not biodegradable under environmental conditions,
but indicates that more work will be necessary to establish
biodegradability.
(ii) If, in a toxicity test containing both the test substance and a ref-
erence compound in the same test vessel, less than 35 percent degradation
(based on total DOC) or less than 25 percent (based on total ThOD or
ThCCh) occurred within 14 days, the test substance can be assumed to
be inhibitory (see paragraph (e)(2) of this guideline for other toxicity tests).
In this case the test series should be repeated, using a lower concentration
of test substance (if this can be done without seriously impairing the accu-
racy of the DOC determination) and/or a higher concentration of inoculum,
but not greater than 30 mg of solids per liter.
(iii) Other conditions for the validity of test results specific to individ-
ual methods are set out under the headings for those tests.
(5) Test report. The test report must include the following:
(i) Test substance. (A) Physical nature and, where relevant, physical/
chemical properties.
(B) Identification data.
(ii) Test conditions. (A) Inoculum: Nature and sampling site(s), con-
centration, and any preconditioning treatment.
(B) Proportion and nature of industrial waste water in sewage, if
known.
(C) Test duration and temperature.
(D) In the case of poorly soluble test substances, methods of prepara-
tion of test solutions/suspensions.
(E) Test method applied; scientific reasons and explanation for any
change of procedure.
(iii) Results. (A) Data in tabular form.
(B) Any observed inhibition phenomena.
(C) Any observed abiotic degradation.
(D) Specific chemical analytical data, if available.
(E) Analytical data on intermediates, if available.
13
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(F) The graph of percent degradation vs. time for the test and ref-
erence substances, the lag phase, the degradation phase, the 10-day win-
dow, and the maximum slope of the degradation curve (see paragraph (c)
of this guideline for definitions).
(G) Percent removal at the plateau, at the end of the test, and/or after
the 10-day window.
(iv) Discussion of results.
(1) DOC die-away test—(1) General. Matters concerning the assess-
ment of biodegradability are discussed under paragraph (j) of this guideline
and it is advisable to read this material before proceeding. For this method,
the test substance should be nonvolatile and have a solubility in water
of at least 100 mg/L. Also, the carbon content and preferably the purity
or relative proportions of major components must be known. This test is
virtually the same as the ISO Standard 7827-1984. It is similar to the
Modified OECD Screening test under paragraph (p) of this guideline but
allows the use of much higher microbial cell densities.
(2) Principle of the test. A measured volume of inoculated mineral
nutrient medium, containing a known concentration of the test substance
(10-40 mg DOC/L) as the nominal sole source of OC, is aerated in the
dark or diffuse light at 22 + 2 °C. Degradation is followed by DOC analysis
at frequent intervals over a 28-day period. The degree of biodegradation
is calculated by expressing the concentration of DOC removed (corrected
for that in the inoculum control) as a percentage of the concentration ini-
tially present. Primary biodegradation may also be calculated from supple-
mental chemical analysis for parent compound made at the beginning and
end of incubation.
(3) Description of the method—(i) Apparatus. In addition to normal
laboratory equipment, the following apparatus should be available:
(A) Conical flasks, e.g. 250-mL to 2-L, depending on the volume
needed for DOC analysis. The flasks must be carefully cleaned with, for
example, alcoholic hydrochloric acid, rinsed, and dried before each test.
(B) Shaking machine—to accommodate the conical flasks, either with
automatic temperature control or used in a constant temperature room—
and of sufficient power to maintain aerobic conditions in all flasks.
(C) Filtration apparatus, with suitable membrane filters.
(D) DOC analyzer.
(E) Apparatus for determining DO, to confirm that the flask contents
are aerobic.
(F) Centrifuge.
14
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(ii) Water. A description of the water to be used is given under para-
graph (j)(2) of this guideline.
(iii) Stock solutions for mineral nutrient medium. Prepare the fol-
lowing stock solutions using analytical grade reagents:
(A) Dissolve 8.50 g potassium dihydrogen orthophosphate
21.75 g dipotassium hydrogen orthophosphate (K^HPCU), 33.40 g
disodium hydrogen orthophosphate dihydrate (Na2HPO4-2H2O), and
0.5 g ammonium chloride (NfUCl) in water and make up to 1 L. The
pH of the solution should be 7.4.
(B) Dissolve 27.50 g calcium chloride, anhydrous (CaCk), or 36.40
g calcium chloride dihydrate, CaCl2-2H2O), in water and make up to
1L.
(C) Dissolve 22.50 g magnesium sulfate heptahydrate (MgSO4-7H2O)
in water and make up to 1 L.
(D) Dissolve 0.25 g iron(III) chloride hexahydrate (FeCl3-6H2O) in
water and make up to 1 L.
(E) In order to avoid having to prepare this solution immediately be-
fore use, add one drop of concentrated HC1 or 0.4 g
ethylenediaminetetraacetic acid (EDTA disodium salt) per liter. If a pre-
cipitate forms in a stock solution replace with a freshly made solution.
(iv) Preparation of mineral nutrient medium. Mix 10 mL of solu-
tion (A) with 800 mL water, then add 1 mL of solutions (B), (C) and
(D) and make up to 1 L with water.
(v) Stock solution of test substance. When the solubility of the sub-
stance exceeds 1 g/L, dissolve 1-10 g, as appropriate, of test or reference
substance in water and make up to 1 L. Otherwise, prepare stock solutions
in mineral nutrient medium or add the chemical directly to the mineral
nutrient medium, making sure it dissolves.
(vi) Inoculum. The inoculum may be derived from a variety of
sources: Activated sludge; sewage effluents; surface waters; soils; or a
mixture of these.
(A) Inoculum from activated sludge. (7) Collect a fresh sample of
activated sludge from the aeration tank of a sewage treatment plant or
laboratory-scale unit treating predominantly domestic sewage. Remove
coarse particles if necessary by filtration through a fine sieve and keep
the sludge aerobic thereafter.
(2) Alternatively, after removal of any coarse particles, settle or cen-
trifuge (e.g. at 1,100 x g for 10 min). Discard the supernatant. The sludge
may be washed in the mineral nutrient medium. Suspend the concentrated
15
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sludge in mineral nutrient medium to yield a concentration of
3-5 g SS/L. Thereafter aerate until required.
(3) Sludge should be taken from a properly working conventional
treatment plant. If sludge has to be taken from a high-rate treatment plant,
or is thought to contain inhibitors, it should be washed. Settle or centrifuge
the resuspended sludge after thorough mixing, discard the supernatant and
again resuspend the washed sludge in a further volume of mineral nutrient
medium. Repeat this procedure until the sludge is considered to be free
from excess substrate or inhibitor.
(4) After complete resuspension is achieved, or with untreated sludge,
withdraw a sample just before use for the determination of the dry weight
of the SS.
(5) A further alternative is to homogenize activated sludge
(3-5 g SS/L). Treat the sludge in a blender for 2 min at medium speed.
Settle the blended sludge for 30 min or longer if required and decant liquid
for use as inoculum at the rate of about 10 mL/L of mineral nutrient me-
dium.
(B) Other sources of inoculum. (7) Alternatively, the inoculum can
be derived from the secondary effluent of a treatment plant or laboratory-
scale unit receiving predominantly domestic sewage. Collect a fresh sam-
ple and keep it aerobic during transport. Allow to settle for 1 h or filter
through a coarse filter paper and keep the decanted effluent or filtrate aero-
bic until required. Up to 100 mL of this type of inoculum may be used
per liter of medium.
(2) Another source for the inoculum is surface water. In this case,
collect a sample of an appropriate surface water (e.g. river, lake) and keep
aerobic until required. If necessary, concentrate the inoculum by filtration
or centrifugation.
(vii) Preconditioning of inoculum. Preconditioning is discussed in
paragraph (j)(6) of this guideline.
(viii) Preparation of flasks. (A) Introduce 800-mL-portions of min-
eral nutrient medium into 2-L conical flasks and add sufficient volumes
of stock solutions of the test and reference substances to separate flasks
to give a concentration of chemical equivalent to 10-40 mg DOC/L. Check
the pH values and adjust to 7.4, if necessary. Inoculate the flasks with
activated sludge or other source of inoculum to give a final concentration
not greater than 30 mg SS/L. Also prepare inoculum controls in the min-
eral nutrient medium but without test or reference substance.
(B) If required, use one vessel to check the possible inhibitory effect
of the test substance by inoculating a solution containing comparable con-
16
-------
centrations of both the test and a reference substance in the same test
vessel.
(C) If required, check whether the test substance is degraded
abiotically by setting up a flask containing a sterilized, uninoculated solu-
tion of the substance. Abiotic controls are discussed in paragraph (j)(7)
of this guideline.
(D) If the test substance is suspected of being significantly adsorbed
onto glass, sludge, etc., make a preliminary assessment to determine the
likely extent of adsorption and thus the suitability of the test for the chemi-
cal (see Table 1, under paragraph (f)(l) of this guideline). Set up a flask
containing the test substance, inoculum and sterilizing agent.
(E) Make up the volumes in all flasks to 1 L with mineral nutrient
medium and, after mixing, take a sample from each flask to determine
the initial concentration of DOC in duplicate (see paragraph (k)(2)(iv) of
this guideline). Cover the openings of the flasks in such a way as to allow
free exchange of air between the flask and the surrounding atmosphere,
for example using aluminum foil. To start the test, place the vessels in
the shaking machine.
(ix) Number of flasks. In a typical run, the following flasks are used:
(A) Flasks 1 and 2—containing test substance and inoculum (test sus-
pension).
(B) Flasks 3 and 4—containing only inoculum (inoculum control).
(C) Flask 5—containing reference compound and inoculum (proce-
dure control).
(D) Flask 6—containing test substance and sterilizing agent (abiotic
sterile control: Optional).
(E) Flask 7—containing test substance, inoculum and sterilizing agent
(adsorption control: Optional).
(F) Flask 8—containing test substance, reference compound and
inoculum (toxicity control: Optional).
(4) Procedure—(i) DOC determinations. Throughout the test, deter-
mine the concentrations of DOC in samples from each flask in duplicate
at known time intervals. It is mandatory to follow DOC in the test suspen-
sion and inoculum blanks in parallel. It is advisable to follow DOC in
the other flasks in parallel as well, although this may not always be pos-
sible.
(ii) Sampling. Take only the minimum volume of test suspension
necessary for each determination. Before sampling make up for any evapo-
ration losses from the flasks by adding water in the required amount. Mix
17
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the culture medium thoroughly before withdrawing a sample and ensure
that material adhering to the walls of the vessels is redissolved or resus-
pended before sampling. Membrane-filter or centrifuge the sample imme-
diately after it has been taken. For other information on sampling, consult
paragraph (k)(2)(iv) of this guideline.
(iii) Frequency of sampling. Ensure that a sufficient number of sam-
ples are collected to allow the percent removal in the 10-day window
to be assessed. No precise sampling pattern can be described. If analyses
are performed on the day of sampling, assess the next sampling time by
considering the result of the analysis. If the samples are preserved, collect
samples daily or every 2 days. Analyze the last samples (28-day) first
and, by a stepwise "backwards" selection of appropriate samples for anal-
ysis, it is possible to obtain a good description of the bio degradation curve
with a relatively small number of determinations. If the last samples
(28-d) show no degradation, no further samples need be analyzed.
(5) Data and reporting — (i) Treatment of results. (A) Data from
the test should be entered on a data sheet, such as the DOC Die-Away
Test Data Sheet following paragraph (1) of this guideline.
(B) The percent degradation (Dt) at each time a sample was taken
should be calculated separately for both flasks containing test substance
(i.e. flasks 1 and 2) using mean values of duplicate DOC measurements
(see data sheet) so that the validity of the test can be assessed (see para-
graph (k)(4) of this guideline). It is calculated using the following equa-
tion:
Dt = [1 - (Q - Cbi(t))/(Co - Cbi(o))] x 100
where:
Dt = percent degradation at time t (days)
Co = mean starting concentration of DOC in the inoculated culture
medium containing test substance (mg DOC/L)
Ct = mean concentration of DOC in the inoculated culture medium
containing test substance at time t (mg DOC/L)
= mean starting concentration of DOC in blank inoculated min-
eral nutrient medium (mg DOC/L)
= mean concentration of DOC blank inoculated mineral nutrient
medium at time t (mg DOC/L)
All concentrations are measured experimentally.
(C) If the test has complied with the validity criteria, display the
course of degradation graphically using the mean of both flasks containing
test substance. Indicate the 10-day window. Calculate and report the per-
18
-------
centage removal achieved at the plateau, at the end of the test and/or at
the end of the 10-day window, as appropriate.
(D) When specific chemical analytical data are available, calculate
percent primary bio degradation (see paragraph (k)(l)(ii) of this guideline).
(E) When an abiotic sterile control is used calculate the percent abi-
otic degradation as follows:
% abiotic degradation = (CS(o> - CS(t))/CS(0) x 100
where:
CS(0) = DOC concentration in sterile control at day 0
CS(t) = DOC concentration in sterile control at day t.
(ii) Validity of tests. The validity criteria apply as given under para-
graph (k)(4) of this guideline.
(iii) Test report. The test report should include the information de-
scribed in under paragraph (k)(5) of this guideline. Data from the test
should be entered on the data sheet. A similar format may be used for
the procedure and toxicity controls.
19
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DOC DIE-AWAY TEST
DATA SHEET
LABORATORY:
DATE AT START OF TEST:
TEST SUBSTANCE:
Name:
Stock solution concentration (mg/L as chemical):
Initial concentration in medium (mg/L as chemical):
INOCULUM:
Source:
Treatment given:
Preconditioning, if any:
SS concentration in reaction mixture (mg/L):
1. CARBON DETERMINATIONS
Carbon analyzer used:
Test substance
plus inoculum
Control: Inoculum
but no test sub-
stance
Flask
no.
1
1
1
2
2
2
3
3
3
4
4
4
3i
32
mean Ca(t)
bi
b2
mean, Cb(t)
Ci
C2
mean, Co(t)
di
d2
mean Cd(t)
mean, Cbi(t> = (C0(t) + Cd(t>)/2
0
DOC aft
ti
er t days
t2
(mg/L)
t3
t4
20
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2. EVALUATION OF RAW DATA
Flask no.
1
2
Mean1
Calculation of results
Di = [1 - (Ca(t) - Cbi(t))/(Ca(o) - Cbi(o))] x 100
D2 = [1 - (Cb(t) - Cbi(t))/(Cb(o) - Cbi(o))] x 100 ....
D, = CD, + DoV2
% degradation after t days
0
0
0
0
ti
t2
t3
t4
and D2 should not be averaged if there is a considerable difference between replicates
3. ABIOTIC DEGRADATION (optional)
DOC concentration in sterile control (mg/L)
Time (
0
days)
t
% abiotic degradation = (CS(o) - CS(t))/CS(o) x 100
4. SPECIFIC CHEMICAL ANALYSIS (optional)
Sterile control
Inoculated test medium
residual amount of test
chemical at end of test
sb
sa
% primary degradation
(Sb - Sa)/Sb x 1 00
21
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(m) Carbon dioxide evolution test — (1) General. Matters concern-
ing the assessment of biodegradability are discussed under paragraph (j)
of this guideline and it is advisable to read this before proceeding. For
this method, the test substance should be nonvolatile and its carbon content
and degree of purity or relative proportions of major components must
be known.
(2) Principle of the test. A measured volume of inoculated mineral
nutrient medium, containing a known concentration of the test substance
(10-20 mg DOC or TOC/L) as the nominal sole source of OC is aerated
by the passage of carbon dioxide-free air at a controlled rate in the dark
or in diffuse light, at 22 + 2 °C. Degradation is followed over 28 days
by determining the CCh produced. The CCh is trapped in Ba(OH)2 or
NaOH and is measured by titration of the residual hydroxide, or as 1C.
The amount of CCh produced from the test substance (corrected for that
derived from the inoculum control) is expressed as a percentage of the
ThCCh. The degree of biodegradation may also be calculated from supple-
mental DOC analysis made at the beginning and end of incubation.
(3) Description of the method — (i) Apparatus. In addition to normal
laboratory equipment, the following apparatus should be available:
(A) Flasks, 2- to 5-L, each fitted with an aeration tube reaching near-
ly to the bottom of the vessel and an outlet (the tube must not interfere
with a magnetic stirrer when used).
(B) Magnetic stirrers, when assessing poorly soluble chemicals.
(C) Gas absorption bottles.
(D) Device for controlling and measuring air flow.
(E) Apparatus for CO2 scrubbing, for preparation of air which is free
from CO2; alternatively, a mixture of CO2-free oxygen and CO2-free nitro-
gen supplied from a gas cylinder in the correct proportions (20% 02: 80%
may be used.
(F) Device for determination of CO2, either titrimetrically or using
some form of 1C analyzer.
(G) Membrane filtration device (optional).
(H) DOC analyzer (optional).
(ii) Water. A description of the water to be used is given under para-
graph (j)(2) of this guideline.
(iii) Stock solutions for mineral nutrient medium. Prepare the same
stock solutions as described under paragraph (l)(3)(iii) of this guideline.
22
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(iv) Preparation of mineral nutrient medium. See paragraph
(l)(3)(iv) of this guideline.
(v) Stock solution of test substance. Prepare stock solutions as de-
scribed in paragraph (l)(3)(v) of this guideline. For the handling of poorly
soluble substances see paragraph (f)(2) of this guideline.
(vi) Inoculum. The inoculum may be derived from a variety of
sources: Activated sludge; sewage effluents; surface waters; soils; or from
a mixture of these.
(vii) Inoculum from activated sludge. See paragraphs
(l)(3)(vi)(A)(7) through (5) of this guideline for details on the collection
and preparation of inoculum from activated sludge.
(viii) Other sources of inoculum. Alternatively, the inoculum can
be derived from the secondary effluent of a treatment plant or laboratory-
scale unit receiving predominantly domestic sewage or from surface water.
See paragraph (l)(3)(vi)(B) of this guideline for details.
(ix) Preconditioning of inoculum. Preconditioning is discussed in
paragraph (j)(6) of this guideline.
(x) Preparation of flasks. (A) As an example, the following volumes
and weights indicate the values for 5-L flasks containing 3 L of inoculated
mineral nutrient medium. If smaller volumes are used modify the values
accordingly, but ensure that the CCh formed can be measured accurately.
To each 5-L flask add 2,400 mL of mineral nutrient medium. Add an
appropriate volume of the prepared activated sludge to give a concentration
of SS of not more than 30 mg/L in the final 3 L of inoculated mixture.
Alternatively, first dilute the prepared sludge to give a suspension of
500-1,000 mg/L in the mineral nutrient medium before adding an aliquot
to the contents of the 5-L flask to attain a concentration of 30 mg/L;
this ensures greater precision (other sources of inoculum may be used;
see paragraph (l)(3)(vi)(B) of this guideline). Aerate these inoculated mix-
tures with CCh-free air overnight to purge the system
(B) Add the test material and reference compound to separate rep-
licate flasks as known volumes of stock solutions, to yield concentrations
of 10 to 20 mg DOC or TOC per liter; leave some flasks without addition
of chemicals as inoculum controls. Add poorly soluble test substances di-
rectly to the flasks on a weight or volume basis or handle as described
under paragraph (f)(2) of this guideline. Make up the volumes of inocu-
lated medium in all flasks to 3 L by the addition of mineral nutrient me-
dium previously aerated with CCh-free air.
(C) If required, use one flask to check the possible inhibitory effect
of the test substance by adding both the test and reference substances to
the same flask at the same concentrations as present in the other flasks.
23
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(D) If required, check whether the test substance is degraded
abiotically by setting up a flask containing a sterilized, uninoculated solu-
tion of the chemical. Abiotic controls are discussed in paragraph (j)(7)
of this guideline.
(E) If Ba(OH)2 is used, connect three absorption bottles, each contain-
ing 100 mL of 0.0125 M Ba(OH)2 solution, in series, to each 5-L flask.
The solution must be free of precipitated sulfate and carbonate and its
concentration must be determined immediately before use. If NaOH is
used, connect two bottles, the second acting as a control to demonstrate
that all of the CCh was absorbed in the first. Absorption bottles fitted
with serum bottle closures are suitable. Add 200 mL of 0.05 M NaOH
to each bottle. This is sufficient to absorb the total quantity of CCh evolved
when the test substance is completely degraded. The NaOH solution, even
when freshly prepared, will contain traces of carbonates; this is corrected
by deduction of the carbonate in the blank control.
(F) Optionally, samples may be withdrawn for analysis of DOC (see
paragraph (k)(2)(iv) of this guideline) and/or specific chemical analysis.
(xi) Number of flasks. In a typical run, the following flasks are used:
(A) Flasks 1 and 2—containing test substance and inoculum (test sus-
pension).
(B) Flasks 3 and 4—containing only inoculum (inoculum control).
(C) Flask 5—containing reference compound and inoculum (proce-
dure control).
(D) Flask 6—containing test substance and sterilizing agent (abiotic
sterile control: Optional).
(E) Flask 7—containing test substance, reference compound and
inoculum (toxicity control: Optional).
(4) Procedure, (i) Start the test by bubbling CO2-free air through
the suspensions at a rate of 30-100 mL/min.
(ii) CCh determinations. (A) It is mandatory to follow the CO2 evo-
lution from the test suspensions and inoculum controls in parallel and it
is advisable to do the same for the other test vessels.
(B) It is recommended that during the first 10 days analyses of CO2
be made every 2nd or 3rd day, and after this at least every 5th day until
the 28th day so that the 10-day window period can be identified.
(C) On the days of CO2 measurement, disconnect the Ba(OH)2 ab-
sorber closest to the test vessel and titrate the hydroxide solution with
0.05 M HC1 using phenolphthalein as the indicator. Move the remaining
absorbers one place closer to the test vessel and place a new absorber
24
-------
containing 100 mL fresh 0.0125 M Ba(OH)2 at the far end of the series.
Perform titrations as needed; for example, when substantial precipitation
is seen in the first bottle and before any is evident in the second. Alter-
natively, with NaOH as absorbent, withdraw a sample of the NaOH solu-
tion from the absorber nearest to the test vessel using a syringe. The sam-
ple volume needed will depend on the carbon analyzer used, but sampling
should not significantly change the absorbent volume over the test period.
Inject the sample into the 1C part of the carbon analyzer for analysis of
evolved CCh directly. Analyze the contents of the second bottle only at
the end of the test in order to correct for any carryover
(D) Optionally, on the 28th day withdraw samples for DOC and/or
specific chemical analysis. Add 1 mL of concentrated HC1 to each test
vessel and aerate overnight to drive off the CO2 present in the test suspen-
sions. On day 29 make the last analysis of evolved CO2.
(5) Data and reporting — (i) Treatment of results. (A) Data from
the test should be entered on a data sheet, such as the CO2 Evolution
Test Data Sheet following paragraph (m) of this guideline.
(B) The amount of CO2 produced is calculated from the amount of
base remaining in the absorption bottle. When 0.0125 M Ba(OH)2 is used
as the absorbent, the amount remaining is assessed by titrating with
0.05 M HC1. Thus 50 mL HC1 is needed to titrate 100 mL of Ba(OH)2.
(C) Since 1 mmol of CO2 is produced for every millimole of Ba(OH)2
reacted to BaCl2 and 2 mmol of HC1 is needed for the titration of the
remaining Ba(OH)2, and given that the molecular weight of CO2 is
44 g/mol, the weight of CO2 produced (given in milligrams) is calculated
as follows:
mg CO2 = [0.05 x (50 - ml HC1 titrated) x 44)/2] =
1. lx (50- ml HC1 titrated)
In this case, the factor to convert volume of HC1 titrated to milligrams
of CO2 produced is 1.1. Calculate the weights of CO2 produced from the
inoculum alone and from the inoculum plus test substance using the re-
spective titration values; the difference is the weight of CO2 produced from
the test substance alone. For example, if the inoculum alone gives a titra-
tion of 48 mL and inoculum plus test substance gives 45 mL,
(a) = CO2 from inoculum = 1. 1 x (50 - 48) = 2.2 mg
(b) = CO2 from inoculum + test substance =
l.lx(50-45) = 5.5mg
and the weight of CO2 produced from the test substance is given by
(a) = 3.3mg.
25
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mg ThCO2
(D) Calculate the weight of ThCCh as follows:
no. of carbons in TS x molecular weight of CO2 x mg of test substance added
molecular weight of test substance
(E) The percent biodegradation is calculated from:
mg CO2 produced
% degradation = x 100
mg ThCO2
or
mg CO2 produced
% degradation = x 100
mg TOC added in test x 3.67
where 3.67 is the conversion factor (44/12) for carbon to CCh. Obtain
the percent degradation after any time interval by adding the percent
ThCCh values calculated for each of the days, up to that time on which
it was measured.
(F) When NaOH is used as the absorbent, calculate the amount of
CO2 produced after any time interval from the concentration of 1C and
the volume of absorbent used. Calculate the percent degradation from:
mg 1C from test flask - mg of 1C from inoculum control
%ofThCO2 = x100
mg TOC added as test substance
(G) Display the course of degradation graphically and indicate the
10-day window. Calculate and report the percentage removal achieved at
the plateau, at the end of the test and/or at the end of the 10-day window,
whichever is appropriate.
(H) When appropriate, calculate DOC removals using the equation
given in paragraph (l)(5)(i)(B) of this guideline.
(I) When an abiotic control is used, calculate the percent abiotic deg-
radation as follows:
mg CO2 produced by sterile flask after 28 days
% abiotic degradation = x 100
mg ThCO2
(ii) Validity of tests. (A) The 1C content of the test suspension at
the beginning of the test must be less than 5 percent of the TC, and the
total CO2 evolution in the inoculum control at the end of the test should
not normally exceed 40 mg/L medium. If values greater than
26
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70 mg CO2/L are obtained, the data and experimental technique should
be examined critically.
(B) The other validity criteria given under paragraph (k)(4) of this
guideline also apply.
(iii) Test report. The test report should include the information de-
scribed under paragraph (k)(5) of this guideline. Data should be entered
on the data sheet.
27
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CO2 Evolution Test
DATA SHEET
LABORATORY:
DATE AT START OF TEST:
TEST SUBSTANCE:
Name:
Stock solution concentration (mg/L as chemical):
Initial concentration in medium (mg/L as chemical):
Total C added to flask (mg C):
ThC02:
INOCULUM:
Source:
Treatment given:
Preconditioning, if any:
SS concentration in reaction mixture (mg/L):
1. CO2 PRODUCTION AND DEGRADABILITY
Method: Ba(OH)2/NaOH/other
Time (days)
0
tj
t2
t3
t4
28
CO2 produced (mg)
test chemical
flask
1
flask
2
inoculum control
flask
3
flask
4
mean
Cumulative
CO2 produced
(mg)
test-control
mean
flask
1
flask
2
% ThCO2 = mg cumu-
lative CO2/mg ThCO2
flask
1
flask
2
mean1
1 Do not take mean if there is a considerable difference between replicates.
NOTE: Similar formats may be used for the procedure control and toxicity
controls (if any).
28
-------
2. CARBON ANALYSIS (optional; see paragraph (l)(5)(i)(B) of this
guideline)
Carbon analyzer used:
Time (days)
0
281
Test chemical (mg/L)
C0
ct
Blank (mg/L)
1 or at end of incubation
% DOC removed = [1 - (Ct - Cbi(t))/C0 - Cbi(o)] x 100
3. ABIOTIC DEGRADATION (optional)
% abiotic degradation =
mg CCh formed in sterile flask after 28 days/mg ThCCh x 100
29
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(n) Modified MITI test (I)—(1) General. Matters concerning the
assessment of biodegradability are discussed under paragraph (j) of this
guideline and it is advisable to read that material before proceeding. For
this method, the formula of the test substance and its purity, or relative
proportions of major components, must be known so that the ThOD may
be calculated. Insoluble and volatile substances may be assessed provided
precautions are taken. Insoluble substances should be dispersed, for exam-
ple, by using very finely ground material or ultrasonics, but not solvents
or emulsifying agents. For volatile substances the volume of "dead" gas
space in the automatic respirometer should be kept to a minimum.
(2) Principle of the test. The oxygen uptake by a stirred solution,
or suspension, of the test substance in a mineral nutrient medium, inocu-
lated with specially grown, unadapted microorganisms, is measured auto-
matically over a period of 28 days in a darkened, enclosed respirometer
at 25 + 1 °C. Evolved CCh is absorbed by soda lime. Biodegradation is
expressed as the oxygen uptake relative to the theoretical uptake (ThOD).
The percentage primary biodegradation is also calculated from supple-
mental specific chemical analysis made at the beginning and end of incu-
bation, and optionally, ultimate biodegradation is determined by DOC
analysis.
(3) Description of the method— (i) Apparatus. In addition to nor-
mal laboratory equipment, the following apparatus should be available:
(A) Automatic electrolytic BOD meter or respirometer normally
equipped with six bottles of 300-mL capacity each, and equipped with
cups to contain CO2 absorbent.
(B) Constant temperature room and/or water bath maintained at
25+1 °C.
(C) Membrane filtration assembly (optional).
(D) Carbon analyzer (optional).
(ii) Water. A description of the water to be used is given under para-
graph (j)(2) of this guideline.
(iii) Stock solutions for mineral nutrient medium. Prepare the fol-
lowing stock solutions using analytical grade reagents:
(A) Dissolve 8.50 g potassium dihydrogen orthophosphate (KH2PO4),
21.75 g dipotassium hydrogen orthophosphate (K.2HPO4), 44.60 g
disodium hydrogen orthophosphate dodecahydrate (Na2HPO4-12H2O), and
1.70 g ammonium chloride (NH4C1) in water and make up to 1 L. The
pH value of the solution should be 7.2
30
-------
(B) Prepare calcium chloride, magnesium sulfate, and iron(III) chlo-
ride solutions as described in paragraphs (l)(3)(iii)(B) through (D) of this
guideline.
(iv) Preparation of mineral nutrient medium. Take 3 mL of each
solution and make up to 1 L.
(v) Stock solution of test substance. Prepare stock solutions in the
mineral nutrient medium or add the chemical directly to the medium.
When the solubility of the test or reference substance exceeds 1 g/L, dis-
solve 1-10 g, as appropriate, of the substance in water and make up to
1 L. For the handling of poorly soluble substances, see paragraph (f)(2)
of this guideline.
(vi) Preparation of inoculum. (A) Collect fresh samples from no
fewer than 10 sites, mainly in areas where a variety of chemicals are used
and discharged. Collect 1-L samples of sludge, surface soil, water, etc.
from sites such as sewage treatment works, industrial wastewater treatment
works, rivers, lakes and seas, and mix thoroughly together.
(B) After removing floating matter and allowing the samples to stand,
adjust the supernatant to pH 7+1 with NaOH or HsPCU. Use an appro-
priate volume of the filtered supernatant to fill a fill-and-draw activated
sludge vessel and aerate the liquid for 23.5 h. Thirty minutes after stopping
aeration, discard about one-third of the whole volume of supernatant and
add an equal volume of a solution (pH 7) containing 0.1 percent each
of glucose, peptone, and potassium orthophosphate to the settled material,
and recommence aeration. Repeat this procedure once per day.
(C) The sludge unit must be operated according to normal sound lab-
oratory practice. Effluents should be clear; temperature should be kept at
25 + 2 °C and pH at 7+ 1; sludge should settle well; there should be suffi-
cient aeration to keep the mixture aerobic at all times; protozoa should
be present; and the activity of the sludge should be tested against a ref-
erence substance at least every 3 months. Do not use sludge as inoculum
until after at least 1 month of operation, but not after more than 4 months.
Thereafter, sample from at least 10 sites at regular intervals, once every
3 months. In order to maintain fresh and old sludge at the same activity,
mix the filtered supernatant of an activated sludge in use with an equal
volume of the filtered supernatant of a freshly collected 10-source mixture
and culture the combined liquor as above. Take sludge for use as inoculum
18-24 h after the unit has been fed.
(vii) Preparation of bottles. Prepare the following six bottles:
(A) Bottle 1—test substance in water at 100 mg/L (abiotic control).
(B) Bottles 2, 3, and 4—test substance in mineral nutrient medium
at 100 mg/L.
31
-------
(C) Bottle 5—reference compound (e.g. aniline) in mineral nutrient
medium at 100 mg/L (procedure control).
(D) Bottle 6—mineral nutrient medium only (inoculum control).
(E) Add poorly soluble test substances directly on a weight or volume
basis, or handle as described under paragraph (f)(2) of this guideline ex-
cept that neither solvent nor emulsifying agent should be used. Add the
062 absorbent to all test vessels in the special cups provided. Adjust the
pH in bottles 2, 3, and 4 to 7.0 before inoculation, if necessary.
(4) Procedure, (i) Inoculate vessels 2, 3, and 4 (test suspensions),
5 (procedure control), and 6 (inoculum control) with a small volume of
the inoculum to give a concentration of 30 mg/L SS. No inoculum is added
to Bottle 1, which serves as an abiotic control. Assemble the equipment,
check that it is air-tight, start the stirrers, and start the measurement of
oxygen uptake under conditions of darkness. Check the temperature, stir-
rer, and coulometric oxygen uptake recorder, and note any changes in color
of the contents of the vessels on a daily basis. Read the oxygen uptake
for the six bottles directly by an appropriate method, for example, from
the 6-point chart recorder, which produces a BOD curve.
(ii) At the end of incubation, normally 28 days, measure the pH of
the contents of the bottles and determine the concentration of the residual
test substance and any intermediates and, in the case of water-soluble sub-
stances, the concentration of DOC (see paragraph (k)(2) of this guideline).
Take special care in the case of volatile substances. If nitrification is antici-
pated, determine nitrate and nitrite ion concentrations, if possible.
(5) Data and reporting—(i) Treatment of results— (A) Data from
the test should be entered on a data sheet, such as the Modified MITI
Test Data Sheet following paragraph (n) of this guideline.
(B) Divide the oxygen uptake (in milligrams) in the presence of test
substance after a given time, corrected for uptake by the inoculum control
after the same time, by the weight of the test substance used (in milli-
grams). This yields the BOD expressed as milligrams of oxygen per milli-
gram of mg test substance (TS), that is,
_ mg O2 uptake with TS - mg O2 uptake by inoculum control
BOD = mg O2/mg TS
mg test substance in bottle
The percent biodegradation is then obtained as follows:
BOD (mg O2/mg substance)
% biodegradation = % ThOD = x 100
ThOD (mg O2/mg substance)
32
-------
(C) For mixtures, calculate the ThOD from the elemental analysis,
as for a single compound. Use the appropriate ThOD (ThODNHs or
ThODNos) according to whether nitrification is absent or complete (see
paragraph (k)(2)(ii) of this guideline). If, however, nitrification occurs but
is incomplete, make a correction for the oxygen consumed by nitrification
calculated from the changes in concentrations of nitrite and nitrate ions
(see paragraph (k)(3) of this guideline).
(D) Calculate the percentage primary biodegradation from loss of spe-
cific (parent) chemical using the equation given under paragraph (k)(l)(ii)
of this guideline. If there has been a loss of test substance in Bottle 1,
(abiotic control), report this and use the concentration of test substance
(Sb) after 28 days in this bottle to calculate percent biodegradation.
(E) When determinations of DOC are made (optional), calculate the
percentage ultimate biodegradation at time t using the equation given under
paragraph (l)(5)(i)(B) of this guideline. If there has been a loss of DOC
in Bottle 1, measuring abiotic removal, use the DOC concentration in this
vessel at day 28 to calculate the percent biodegradation.
(ii) Validity of results. (A)The oxygen uptake of the inoculum con-
trol is normally 20-30 mg O2/L and should not be greater than
60 mg O2/L in 28 days. Values higher than 60 mg/L require critical exam-
ination of the data and the experimental technique. If the pH value is out-
side the range 6-8.5 and the oxygen consumption by the test substance
is less than 60 percent, the test could be repeated with a lower concentra-
tion of test substance.
(B) A test is considered valid if the greatest difference between rep-
licate values of the removal of the test substance at the plateau or at the
end of the test, as appropriate, is less than 20 percent, and if the percent
degradation of aniline calculated from the oxygen consumption exceeds
40 percent after 7 days and 65 percent after 14 days. If either of these
conditions is not met, the test should be repeated. Low values do not nec-
essarily mean that the test substance is not biodegradable under environ-
mental conditions, but indicates that more work will be necessary to estab-
lish biodegradability.
(iii) Test report. The test report should include the information out-
lined under paragraph (k)(5) of this guideline. Data from the test should
be entered on the data sheet. If a BOD curve (BOD vs. time) is available,
it should be attached to the data sheet.
33
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MODIFIED MITI TEST
DATA SHEET
LABORATORY:
DATE AT START OF TEST:
TEST SUBSTANCE:
Name:
Stock solution concentration (mg/L as chemical):
Initial concentration in medium (mg/L as chemical):
Volume of reaction mixture, V (mL):
ThOD (mg O2/L):
INOCULUM:
Sludge sampling sites:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Concentration of SS in activated sludge after acclimation with syn-
thetic sewage (mg/L):
Volume of sludge added per liter of final medium (mL):
Concentration of sludge in final medium (mg/L):
34
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1. OXYGEN UPTAKE: BIODEGRADABILITY
Type of respirometer:
O2 uptake
O2 uptake
Corrected
BOD (mg
with test substance (mg)
3i
82
a3
by inoculum control (mg)
b
O2 uptake (mg)
ai - b
a2 - b
83 — b
O2/mg test substance)
ai - b/C0V
a2 - b/C0V
a, - b/CnV
% degradation (BOD/ThOD x 100)
1
2
3
mean1
Time (days)
ti
t2
t3
tx
1 Do not take a mean if there are considerable differences between replicates.
Note: Similar formats may be used for the other controls.
2. CARBON ANALYSIS: (optional)
Carbon analyzer used:
Flask
Sludge + test substance
Sludge + test substance
Sludge + test substance
Inoculum control
Water + test substance Cabiotic control}
DOC
Measured
3i
a2
a3
b
c
Corrected
ai - b
a2 - b
a3 - b
%
DOC
re-
moved
-
Mean
n
-
% DOC removed = c - (a - b)/c x 100
35
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3. SPECIFIC CHEMICAL ANALYSIS
blank test with water (abiotic control)
inoculated medium
residual amount of test sub-
stance at end of test
Sb
Sal
Sa2
Sa3
% primary degradation
% degradation = Sb - Sa/Sb x 100
Calculate percent primary degradation for bottles ai, a2, and as, respec-
tively.
36
-------
(o) Closed bottle test—(1) General. Matters concerning the assess-
ment of biodegradability are discussed under paragraph (j) of this guideline
and it is advisable to read this before proceeding. For this method, the
formula of the substance and its purity, or relative proportions of major
components, must be known so that the ThOD may be calculated. If the
ThOD cannot be calculated the COD should be determined, but falsely
high values of percent biodegradation may be obtained if the test substance
is incompletely oxidized in the COD test. Insoluble and volatile substances
may be assessed provided that precautions are taken. Degradation values
for insoluble substances may be falsely low unless the bottles are agitated
periodically during the incubation.
(2) Principle of the test. The solution of the test substance in mineral
nutrient medium, usually at 2-5 mg/L, is inoculated with a relatively small
number of microorganisms from a mixed population and kept in com-
pletely full, closed bottles in the dark at constant temperature (22 + 2 °C).
Degradation is followed by analysis of DO over a 28-day period. The
amount of oxygen taken up by the microbial population during biodegrada-
tion of the test substance, corrected for uptake in the inoculum control
run in parallel, is expressed as a percentage of ThOD or, less satisfactorily,
COD.
(3) Description of the method—(i) Apparatus. In addition to normal
laboratory equipment, the following apparatus should be available:
(A) BOD bottles with glass stoppers; e.g. 250- to 300-mL or 100-
to 125-mL. It is important that the bottles are thoroughly cleaned before
use. If the Winkler method for determining DO is used, it is sufficient
to rinse the bottle several times with tap water followed by deionized
water. However, if the electrode method is used, a more stringent cleaning
procedure is required. Add to the empty bottle 5-10 mL of a wash solution
(e.g. 2.5 g iodine plus 12.5 g KI per liter of 1 percent w/v H2SO4), shaking
well to coat the bottle walls. Allow to stand for 15 min, pour off the
solution, and rinse thoroughly with tap water and finally deionized water.
(B) Water bath or incubator for keeping bottles at constant tempera-
ture with the exclusion of light.
(C) Large glass bottles (2- to 5-L) for the preparation of media and
filling the BOD bottles.
(D) Oxygen electrode and meter, or equipment and reagents for
Winkler titration.
(ii) Water. A description of the water to be used is given under para-
graph (j)(2) of this guideline.
37
-------
(iii) Stock solutions for mineral nutrient medium. Prepare the stock
solutions given under paragraph (l)(3)(iii) of this guideline, using analyt-
ical grade reagents.
(iv) Preparation of mineral nutrient medium. Add 1 mL of solu-
tions (A), (B), (C) and (D) as prepared according to paragraph (l)(3)(iii)
of this guideline to 800 mL water, mix, and then make up to 1 L.
(v) Stock solution of test substance. If the solubility of test or ref-
erence substance exceeds 1 g/L, dissolve 1-10 g, as appropriate, in water
and make up to 1 L. Otherwise, prepare stock solutions in mineral nutrient
medium or add the chemical directly to the nutrient medium making sure
that the chemical dissolves.
(vi) Inoculum. (A) The inoculum is normally derived from the sec-
ondary effluent of a treatment plant or laboratory-scale unit receiving pre-
dominantly domestic sewage. Collect and handle as described under para-
graph (l)(3)(vi)(B)(7) of this guideline. Normally use from one drop
(0.05 mL) to 5 mL of filtrate per liter of medium; trials may be needed
to discover the optimum volume for a given effluent.
(B) An alternative source for the inoculum is surface water. In this
case, collect a sample of an appropriate surface water, e.g. river or lake
water, and keep aerobic until required. As with effluents, the optimum
volume to be used as inoculum may have to be determined by trial tests.
(vii) Preconditioning of inoculum. If required, the inoculum may
be preconditioned by aerating the secondary effluent, without other treat-
ment or addition, for 5-7 days at the test temperature.
(viii) Preparation of bottles. (A) Strongly aerate mineral nutrient
medium for at least 20 min and allow to stand. Generally, the medium
is ready for use after standing for 20 h at the test temperature. Carry out
each test series with medium derived from the same batch. Determine the
concentration of DO for control purposes; the value should be about
9 mg/L at 20 °C. Conduct all transfer and filling operations of the air-
saturated medium bubble-free, for example, by the use of siphons.
(B) Prepare parallel groups of BOD bottles for the determination of
the test and reference substances in simultaneous experimental series. As-
semble a sufficient number of BOD bottles, including inoculum controls,
to allow at least duplicate measurements of oxygen consumption to be
made at the desired test intervals, for example, after 0, 7, 14, 21 and 28
days. To ensure that the 10-day window can be identified, more bottles
than this will probably be required.
(C) Add fully-aerated mineral nutrient medium to large bottles so that
they are about one-third full. Then add sufficient amounts of the stock
solutions of the test and reference substances or add by other means (see
38
-------
paragraph (f)(2) of this guideline) to separate large bottles so that the final
concentration of the chemicals is not greater than 10 mg/L (see paragraph
(o)(3)(viii)(D) of this guideline). Do not add test or reference chemical
to the inoculum control medium set up in a separate large bottle.
(D) In order to ensure that the inoculum activity is not limited, the
concentration of DO must not fall below 0.5 mg/L in the BOD bottles.
Generally, this limits the concentration of test substance to about
2 mg/L. An idea of the highest concentration to be used can be obtained
from the ThOD (mg O2/mg chemical) of the test substance. For poorly
degradable compounds and those with a low ThOD, 5-10 mg/L can be
used. In some cases, it would be advisable to run parallel series of test
substance at two different concentrations, for example, 2 and 5 mg/L. Nor-
mally, calculate the ThOD on the basis of formation of ammonium salts,
but if nitrification is expected or known to occur, calculate on the basis
of the formation of nitrate (i.e. ThODNo3); (see paragraph (k)(2)(ii) of
this guideline). However, if nitrification is not complete but does occur,
correct for the changes in concentration of nitrite and nitrate as determined
by analysis (see paragraph (k)(3) of this guideline)
(E) If the toxicity of the test substance is to be investigated (in the
case, for example, of a low biodegradability value having been found pre-
viously), another series of bottles is necessary. Prepare another large bottle
to contain aerated mineral nutrient medium (to about one-third of its vol-
ume) plus test and reference substances at final concentrations the same
as those in the other large bottles.
(F) Inoculate the solutions in the large bottles with secondary effluent
(one drop, or about 0.05 mL, to 5 mL/L (see paragraph (o)(3)(vi)(A) of
this guideline) or with another source such as river water (see paragraph
(o)(3)(vi)(B) of this guideline). Finally, make up the solutions to volume
with aerated mineral nutrient medium using a hose that reaches down to
the bottom of the bottle to achieve adequate mixing.
(ix) Number of bottles. (A) In a typical run the following bottles
are used:
(7) At least 10 containing test substance and inoculum (test suspen-
sion).
(2) At least 10 containing only inoculum (inoculum control).
(3) At least 10 containing reference compound and inoculum (proce-
dure control).
(4) When necessary, six bottles containing test and reference sub-
stances, and inoculum (toxicity control).
(B) About twice as many bottles are required to ensure the ability
to identify the 10-day window as are needed otherwise.
39
-------
(4) Procedure, (i) Dispense each prepared solution or suspension im-
mediately into the respective group of BOD bottles by hose from the lower
quarter (not the bottom) of the appropriate large bottle, so that all the
BOD bottles are completely filled. When testing poorly soluble substances,
added by methods described in paragraph (f)(2) of this guideline, ensure
that the contents of the large bottles are well mixed by stirring. Tap gently
to remove any air bubbles.
(ii) Analyze the O-time bottles immediately for DO by the Winkler
or electrode method. The contents of the bottles can be preserved for later
analysis by the Winkler method by adding MnSO4 and NaOH (the first
Winkler reagent). Store the carefully stoppered bottles, containing the oxy-
gen fixed as brown hydrated manganese(III) oxide, in the dark at
10-20 °C for no longer than 24 h before proceeding with the remaining
steps of the Winkler method. Stopper the remaining replicate bottles ensur-
ing that no air bubbles are enclosed, and incubate at 20 °C in the dark.
(iii) Each series must be accompanied by a complete parallel series
for the determination of the inoculum control. Withdraw at least duplicate
bottles of all series for DO analysis at time intervals (at least weekly)
over the 28 days incubation. Weekly samples should allow the assessment
of percent removal in a 14-day window, whereas sampling every 3-4 days
should allow the 10-day window to be identified, and will require about
twice as many bottles.
(iv) For nitrogen-containing test substances, corrections for uptake of
oxygen by any nitrification occurring should be made. To do this, use
the O2-electrode method for determining the concentration of DO and then
withdraw a sample from the BOD bottle for analysis for nitrite and nitrate.
From the increase in concentration of nitrite and nitrate, calculate the oxy-
gen used (see paragraph (k)(3) of this guideline)
(5) Data and reporting—(i) Treatment of results. (A) Data from
the test should be entered on a data sheet, such as the Closed Bottle Test
Data Sheet followimg paragraph (o) of this guideline.
(B) (7) First calculate the BOD exerted after each time period by
subtracting the oxygen depletion (mg O2/L) of the inoculum control from
that exhibited by the test substance. Divide this corrected depletion by
the concentration (mg/L) of the test substance, to obtain the specific BOD
as milligrams of oxygen per milligrams of test substance:
(mg O2/L uptake by TS - mg O2/L uptake by inoculum control) mg O2
BOD
mg TS/L in bottle mg TS
(2) Calculate the percent biodegradation by dividing the specific BOD
by the specific ThOD (calculated according to paragraph (k)(2)(ii) of this
40
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guideline) or COD (determined by analysis; see paragraph (k)(2)(iii)), as
follows:
BOD (mg O2/mg TS)
% degradation = x 100
ThOD or COD (mg O2/mg TS)
(C) It should be noted that the ThOD and COD methods do not nec-
essarily give the same value; it is preferable to use the former method.
(D) For test substances containing nitrogen, use the appropriate ThOD
(NH4 or NO3) according to what is known or expected about the occur-
rence of nitrification (see paragraph (k)(2)(ii) of this guideline). If nitrifica-
tion occurs but is not complete, calculate a correction for the oxygen
consumed by nitrification from the changes in concentration of nitrite and
nitrate during the 28 days of the test (see paragraph (k)(3) of this guide-
line).
(ii) Validity of results. (A) Oxygen depletion in the inoculum control
should not exceed 1.5 mg DO/L after 28 days. Values higher than this
require investigation of the experimental techniques. The residual con-
centration of oxygen in the test bottles should not fall below 0.5 mg/L
at any time. Such low oxygen levels are acceptable only if the method
used for determining DO is sufficiently accurate.
(B) The other validity criteria given under paragraph (k)(4) of this
guideline also apply.
(iii) Test report. The test report should include the information de-
scribed under paragraph (k)(5) of this guideline. Data from the test should
be entered on the data sheet.
41
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CLOSED BOTTLE TEST
DATA SHEET
LABORATORY:
DATE AT START OF TEST:
TEST SUBSTANCE:
Name:
Stock solution concentration (mg/L as chemical):
Initial concentration in bottle (mg/L as chemical):
ThOD or COD (mg O2/mg TS):
INOCULUM:
Source:
Treatment given:
Preconditioning, if any:
Concentration in reaction mixture (mg/L):
1. DO DETERMINATION
Method: Winkler/electrode
Control — with inoculum but with-
out TS
TS plus inoculum
1
2
mean
1
2
Ci
Qz
nib = Ci + C2/2
3i
a2
m
0
3 O2/L al
ti
ter t day
t2
s
tx
Note: Similar formats may be used for the procedure control and the tox-
icity controls, if any.
42
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2. CORRECTION FOR NITRIFICATION (see paragraph (k)(3) of this
guideline)
(i) Concentration of nitrate (mg N/L)
(ii) Change in nitrate concentration (mg N/L)
(iii) Oxygen equivalent (mg/L)
(iv) Concentration of nitrite (mg N/L)
(v) Change in nitrite concentration (mg N/L)
(vi) Oxygen equivalent (mg/L)
Ciii+vH Total oxvaen eauivalent Cma/D
0
Time of
ti
incubation
t2
(days)
ts
tx
3. DO DEPLETION: % DEGRADATION (% D)
DO depletion after t days (mg/L)
(mb — ai)^
(mb - a2)1
% Dai = (mb - ai)VTS (mg/L) x ThOD x 100
% Da2 = (mb - a2)VTS (mg/L) x ThOD x 100
% D™ 2 = Dai + Da^/2
ti
t2
ts
tx
1 This assumes that mb(0) = ai(0) = a2(0), where mb(0) = blank value at day 0; ai(0) = test substance
value at day 0 in Bottle 1; a2(0) = test substance value at day 0 in Bottle 2. If mb(0) does not equal ai(0)
or a2(o), use [(ai(0) - ai(x)) - (mb(o) - mb(x))] and [(a2(0) - a2(x)) - (mb(0) - mb(X))], where mb(X) = mean
blank value at day x, ai(x) = TS value at day x in Bottle 1, and a2(x> = TS value at day x in Bottle 2.
2 Do not take a mean if there are considerable differences between replicates.
4. INOCULUM CONTROL DO DEPLETIONS:
Oxygen consumption by control = (nib(o) - nib(28)) mg/L.
This consumption is important for the validity of the test and should be
less than 1.5 mg/L. Apply any correction for nitrification.
43
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(p) Modified OECD screening test—(1) Introduction. Matters of
general interest concerning the assessment of biodegradability are dis-
cussed under paragraph (j) of this guideline, and it is advisable to read
this before proceeding. For this method, the test substance should be non-
volatile and have a solubility in water of at least 100 mg/L. Also, the
carbon content and, preferably, the purity or relative proportions of major
components must be known. This method is similar to the DOC die-away
test under paragraph (1) of this guideline but employs a relatively low con-
centration of microorganisms.
(2) Principle of the test. A measured volume of mineral nutrient me-
dium containing a known concentration of the test substance
(10-40 mg DOC/L) as the nominal sole source of OC is inoculated with
0.5 mL effluent per liter of medium. The mixture is aerated in the dark
or diffuse light at 22 + 2 °C. Degradation is followed by DOC analysis
at frequent intervals over a 28-day period. The degree of biodegradation
is calculated by expressing the concentration of DOC removed (corrected
for that in the inoculum control) as a percentage of the concentration ini-
tially present. Primary biodegradation may also be calculated from supple-
mental chemical analysis for the parent compound made at the beginning
and end of incubation.
(3) Description of the method—(i) Apparatus. In addition to normal
laboratory equipment, the following apparatus should be available:
(A) Conical flasks, e.g. 250-mL to 2-L, depending on the volume
needed for DOC analysis. The flasks must be carefully cleaned with e.g.
alcoholic hydrochloric acid, rinsed, and dried before each test.
(B) Shaking machine to accommodate the conical flasks, either with
automatic temperature control or used in a constant temperature room, and
of sufficient power to maintain aerobic conditions in all flasks.
(C) Filtration apparatus, with suitable membranes.
(D) DOC analyzer.
(E) Apparatus for determining DO, to confirm that the flask contents
are aerobic.
(F) Centrifuge.
(ii) Water. A description of the water to be used is given under para-
graph (j)(2) of this guideline.
(iii) Stock solutions for mineral nutrient medium. Prepare the same
stock solutions as described in paragraph (l)(3)(iii) of this guideline, using
analytical grade reagents.
44
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(iv) Preparation of mineral nutrient medium. (A) Prepare medium
as described in paragraph (l)(3)(iv) of this guideline.
(B) The OECD screening test uses only 0.5 mL effluent/L as
inoculum and therefore the medium may need to be fortified with trace
elements and growth factors. This is done by adding 1 mL each of the
following solutions per liter of final medium:
(7) Trace element solution. Dissolve the following salts in water
in a 1-L flask: 39.9 mg manganese sulfate tetrahydrate, MnSO4-4H2O; 57.2
mg boric acid, HsBOs; 42.8 mg zinc sulfate heptahydrate, ZnSCU-TtbO;
34.7 mg ammonium heptamolybdate (NH4)6Mo7O24; and 100.0 mg
iron(III)-chelate, (Fe[ethylenediaminetetraacetic acid]Cls) Make up to 1 L
with water.
(2) Vitamin solution. Dissolve 15.0 mg of yeast extract in 100 mL
of water. Sterilize by passage through a 0.2 (im pore-size membrane, or
prepare immediately before use.
(v) Stock solution of test substance. Prepare stock solution as de-
scribed in paragraph (l)(3)(v) of this guideline.
(vi) Inoculum. The inoculum is derived from the secondary effluent
of a treatment plant or laboratory-scale unit receiving predominantly do-
mestic sewage and should be prepared as described in paragraph
(l)(3)(vi)(B) of this guideline. Use 0.5 mL of the filtrate per liter of me-
dium.
(vii) Preconditioning of inoculum. If required, the inoculum may
be preconditioned as described in paragraph (j)(6) of this guideline.
(viii) Preparation of flasks. (A) Introduce 800-mL-portions of min-
eral nutrient medium into 2-L conical flasks and add sufficient volumes
of stock solutions of the test and reference substances to separate flasks
to give a concentration of chemical equivalent to 10-40 mg DOC/L. Check
the pH value and adjust to pH 7.4 if necessary. Inoculate the flasks with
sewage effluent at 0.5 mL/L medium. Prepare inoculum controls in the
mineral nutrient medium but without test or reference substance.
(B) Toxicity, abiotic, and adsorption controls can be set up, if re-
quired, by following the same procedures as described under paragraphs
(l)(3)(viii)(B) through (D) of this guideline.
(C) Make up the volumes in all flasks to 1 L with mineral nutrient
medium and, after mixing, take a sample from each flask to determine
the initial concentration of DOC in duplicate (see paragraph (k)(2)(iv) of
this guideline). Cover with aluminium foil or other material in such a way
as to allow free exchange of air between the flask and the surrounding
atmosphere, then place the vessels in the shaking machine to start the test.
45
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(ix) Number of flasks. In a typical run, the following flasks are used:
(A) Flasks 1 and 2—containing test substance and inoculum (test sus-
pension).
(B) Flasks 3 and 4—containing only inoculum (inoculum control).
(C) Flask 5 containing reference compound and inoculum (procedure
control).
(D) Flask 6—containing test substance and sterilizing agent (abiotic
sterile control: Optional).
(E) Flask 7—containing test substance, inoculum, and sterilizing
agent (adsorption control: Optional).
(F) Flask 8—containing test substance, reference compound and
inoculum (toxicity control: Optional).
(4) Procedure. See paragraph (1)(4) of this guideline.
(5) Data and reporting. See paragraph (1)(5) of this guideline.
(q) Manometric respirometry test—(1) General. Matters concern-
ing the assessment of biodegradability are discussed under paragraph (j)
of this guideline and it is advisable to read this before proceeding. For
this method, the formula of the test substance and its purity, or relative
proportions of major components, must be known so that the ThOD may
be calculated. If the ThOD cannot be calculated, the COD should be deter-
mined, but falsely high values of percentage biodegradation may be ob-
tained if the test substance is incompletely oxidized in the COD test. Insol-
uble and volatile substances may be assessed provided precautions are
taken.
(2) Principle of the test. A measured volume of inoculated mineral
nutrient medium, containing a known concentration of test substance
(100 mg TS/L giving at least 50-100 mg ThOD/L) as the nominal sole
source of OC, is stirred in a closed flask at a constant temperature
(+ 1 °C or better) for up to 28 days. The consumption of oxygen is deter-
mined either by measuring the quantity of oxygen (produced electro-
lytically) required to maintain constant gas volume in the respirometer
flask, or from the change in volume or pressure (or a combination of the
two) in the apparatus. Evolved CO2 is absorbed in a solution of potassium
hydroxide or other suitable absorbent. The amount of oxygen taken up
by the microbial population during biodegradation of the test substance
(corrected for uptake by the inoculum control, run in parallel) is expressed
as a percentage of ThOD or, less satisfactorily, COD. Optionally, primary
biodegradation may also be calculated from supplemental specific chemi-
cal analysis made at the beginning and end of incubation, and ultimate
biodegradation may be determined by DOC analysis.
46
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(3) Description of the method—(i) Apparatus. In addition to normal
laboratory equipment, the following apparatus should be available:
(A) Suitable respirometer.
(B) Apparatus to maintain temperature at 22 + 2 °C or better.
(C) Membrane filtration assembly (optional).
(D) Carbon analyzer (optional).
(ii) Water. A description of the water to be used is given under para-
graph (j)(2) of this guideline.
(iii) Stock solutions for mineral nutrient medium. Prepare the same
stock solutions as described in paragraph (l)(3)(iii) of this guideline, using
analytical grade reagents.
(iv) Preparation of mineral nutrient medium. Prepare medium as
described in paragraph (l)(3)(iv) of this guideline.
(v) Stock solutions of test substances. Prepare stock solutions as
described in paragraph (l)(3)(v) of this guideline.
(vi) Inoculum. The inoculum may be derived from any of a variety
of sources. See paragraph (l)(3)(vi) for details. If the inculum is derived
from the secondary effluent of a treatment plant or laboratory-scale unit
receiving predominantly domestic sewage, (see paragraph (l)(3)(vi)(B) of
this guideline), use only 0.5 mL of the filtrate per liter of medium.
(vii) Preconditioning of inoculum. If required, the inoculum may
be preconditioned as described in paragraph (j)(6) of this guideline.
(viii) Preparation of flasks. (A) Prepare solutions of the test and
reference substances in separate batches in mineral nutrient medium at 100
mg chemical/L (giving 50-100 mg ThOD/L), using the stock solutions.
Calculate the ThOD on the basis of formation of ammonium salts unless
nitrification is anticipated; in this case, the calculation should be based
on nitrate formation (see paragraph (k)(2)(ii) of this guideline). Determine
the pH values and if necessary adjust to 7.4 + 0.2. Poorly soluble sub-
stances should be added at a later stage.
(B) If the toxicity of the test substance is to be determined, prepare
a solution in mineral nutrient medium containing both test and reference
substances at the same concentrations as in the individual solutions.
(C) If measurement of abiotic degradation is required, prepare a solu-
tion of the test substance in mineral nutrient medium at
100 mg ThOD/L, and sterilize by adding a toxic substance at an appro-
priate concentration. Abiotic controls are discussed in paragraph (j)(7) of
this guideline.
47
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(D) Introduce the requisite volume of solutions of test and reference
substances into at least duplicate respirometer flasks. Add to additional,
separate flasks mineral nutrient medium only (for inoculum controls) and,
if required, the mixed test/reference substance solution and sterile solution
of test substance. If the test substance is poorly soluble, add it directly
at this stage on a weight or volume basis or handle it as described in
paragraph (f)(2) of this guideline. Add potassium hydroxide, soda-lime pel-
lets, or other absorbent to the CCh-absorber compartments.
(ix) Number of flasks. In a typical run the following flasks are used:
(A) Flasks 1 and 2—containing test substance and inoculum (test sus-
pension).
(B) Flasks 3 and 4—containing only inoculum (inoculum control).
(C) Flask 5—containing reference compound and inoculum (proce-
dure control).
(D) Flask 6—containing test substance and sterilizing agent (abiotic
sterile control).
(E) Flask 7—containing test and reference substances, and inoculum
(toxicity control).
(4) Procedure, (i) Allow the vessels to reach the desired temperature
and inoculate appropriate vessels with activated sludge or other inoculum
to give a concentration of SS not greater than 30 mg/L. Assemble the
equipment, start the stirrer, check that the equipment is air-tight, and start
the measurement of oxygen uptake. Usually no further attention is required
other than taking the necessary readings and making daily checks to see
that the correct temperature and adequate stirring are maintained.
(ii) When an automatic respirometer is used, a continuous record of
oxygen uptake is obtained so that the 10-day window is easily recognized.
For nonautomatic respirometers daily readings will be adequate.
(iii) Calculate the oxygen uptake from the readings taken at regular
and frequent intervals, using the methods given by the manufacturer of
the equipment. At the end of incubation, normally 28 days, measure the
pH of the contents of the flasks, especially if oxygen uptake is low or
greater than ThODNH4, for nitrogen-containing chemicals.
(iv) If required, withdraw samples from the respirometer flasks ini-
tially and at the end of the experiment, for analysis of DOC (see paragraph
(k)(2)(iv) of this guideline) and/or specific chemical. At the initial with-
drawal, ensure that the volume of test suspension remaining in the flask
is known. When oxygen is taken up by a nitrogen-containing test sub-
stance, determine the increase in concentration of nitrite and nitrate over
48
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28 days and calculate the correction for the oxygen consumed by nitrifica-
tion (see paragraph (k)(3) of this guideline).
(5) Data and reporting—(i) Treatment of results. (A) Data should
be entered on a data sheet, such as the Manometric Respirometry Test
Data Sheet following paragraph (q) of this guideline.
(B) (7) Calculate the BOD (mg Ch/mg test chemical) exerted after
each time period by dividing the oxygen uptake (in milligrams) in the
presence of the test chemical, corrected for uptake by the inoculum control,
by the weight of the test chemical used in milligrams. That is,
mg O2 uptake with TS - mg O2 uptake by inoculum control
BOD =
mg TS in vessel
(2) Calculate the percent biodegradation as follows:
BOD (mg O2/mg TS)
% degradation = x 100
ThOD or COD (mg O2/mg TS)
(C) When optional determinations of specific chemical and/or DOC
are made, calculate the percent degradation as described in paragraphs
(k)(l)(ii) and (l)(5)(i)(B) of this guideline, respectively.
(ii) Validity of results. (A) The oxygen uptake of the inoculum con-
trol is normally 20-30 mg O2/L and should not be greater than
60 mg/L in 28 days. Values higher than 60 mg/L require critical examina-
tion of the data and experimental technique. If the pH value is outside
the range 6-8.5 and the oxygen consumption by the test substance is less
than 60 percent, the test should be repeated with a lower concentration
of test substance.
(B) The validity criteria given in paragraph (k)(4) of this guideline
also apply.
(iii) Test report. The test report should include the information de-
scribed in paragraph (k)(5) of this guideline. Data should be entered on
the data sheet.
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MANOMETRIC RESPIROMETRY TEST
DATA SHEET
LABORATORY:
DATE AT START OF TEST:
TEST SUBSTANCE:
Name:
Stock solution concentration (mg/L as chemical):
Initial concentration in medium (mg/L as chemical):
Volume in test flask, V (mL):
ThOD or COD (mg O2/mg TS (NH3, NO3):
INOCULUM:
Source:
Treatment given:
Preconditioning, if any:
SS concentration in reaction mixture (mg/L):
50
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1. OXYGEN UPTAKE, BIODEGRADABILITY
Type of respirometer:
O2 uptake with test substance (mg)
O2 uptake by inoculum control (mg)
Corrected O2 uptake (mg)
BOD (mg O2/mg TS)
% degradation, D (BOD/ThOD x 100) ...
3i
a2
bi
b2
(ai - bmean)/mg TS
(a2 - bmean)/mg TS
mean1
ti
Ti
t2
ne (days
t3
)
t4
tx
i and D2 should not be averaged if there is a considerable difference between the values.
Note: Similar formats may be used for the other controls.
2. CORRECTION FOR NITRIFICATION (see paragraph (k)(3) of this
guideline)
(i)
(ii)
(iii)
(iv)
(ii + iv)
concentration of nitrate (mg N/L)
oxygen equivalent (4.57 x N x V) (mg)
concentration of nitrite (mg N/L)
oxygen equivalent (3 43 x N x V) (mg)
total oxvaen eauivalent
Time
0
of incubation (c
28
lays)
difference
(N)
(N)
51
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3. CARBON ANALYSIS (optional; see paragraph (l)(5)(i)(B))
Time (days)
0
281
Test chemical (mg/L)
C0
ct
Inoculum control (mg/L)
1 or at end of incubation
% DOC removed = [1 - (Ct - Cbi(t)/C0 - Cbi(o))] x 100
4. SPECIFIC CHEMICAL ANALYSIS (optional)
Sterile control
Inoculated test medium
residual amount of TS at end of
test
sb
sa
% primary degradation
(Sb - Sa)/Sb x 1 00
5. ABIOTIC DEGRADATION (optional)
a = 62 consumption in sterile flasks at end of test (mg)
a/mg TS = 62 consumption per milligram test chemical
a
% abiotic degradation
x 100
mg TS x ThOD
52
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(r) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Reynolds, L. et al. Evaluation of the toxicity of substances to
be assessed for biodegradability. Chemosphere 16:2259 (1987).
(2) Boethling, R. Biodegradation testing of insoluble chemicals. Envi-
ronmental Toxicology and Chemistry 3:5 (1984).
(3) de Morsier A. et al. Biodegradation tests for poorly soluble com-
pounds. Chemosphere 16:833 (1987).
(4) Draft ISO Standard 10634. Water Quality, Evaluation in an aque-
ous medium of the "ultimate" biodegradability of low-soluble organic
compounds (1990).
(5) Gerike P. The Biodegradability testing of poorly water soluble
compounds. Chemosphere 13:169 (1984).
(6) American Public Health Association. Standard Methods for the
Examination of Water and Wastewater, 12th ed, American Water Pollution
Control Federation, Oxygen Demand, p 65 (1965).
(7) Deutsche Einheitsverfahren zur Wasser, Abwasser und
Schlammuntersuchung, Summarische Wirkungs- und Stoffkenngrossen
(Gruppe H). DIN 38 409 Teil 41. Bestimmung des Chemischen
Sauerstofbedarfs (CSB) (H 41), Normenausschuss Wasserwesen (NAW)
in DIN Deutsches Institut fur Normung e.V.
(8) Wagner R. Vom Wasser 46:139 (1976).
(9) Kelkenberg, H. Zur Wasser und Abwasserforschung 8:146 (1975).
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