United States Prevention, Pesticides EPA712-C-08-003
Environmental Protection And Toxic Substances October 2008
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3215
Inherent
Biodegradability-
Concawe Test
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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
(OPPTS), United States Environmental Protection Agency for use in the testing
of pesticides and toxic substances, and the development of test data to meet the
data requirements of the Agency under the Toxic Substances Control Act (TSCA)
(15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodenticide Act
(FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and
Cosmetic (FFDCA) (21 U.S.C. 346a).
OPPTS developed this guideline through a process of harmonization of
the testing guidance and requirements that existed for the Office of Pollution
Prevention and Toxics (OPPT) in Title 40, Chapter I, Subchapter R of the Code
of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) in
publications of the National Technical Information Service (NTIS) and in the
guidelines published by the Organization for Economic Cooperation and
Development (OECD).
For additional information about OPPTS harmonized guidelines and to
access this and other guidelines, please go to http://www.epa.gov/oppts and
select "Test Methods & Guidelines" on the left side menu.
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OPPTS 835.3215 - Inherent biodegradability - Concawe test
(a) Scope—(1) Applicability. This guideline is intended for use in testing
pursuant to the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. This method is based on International Organization for
Standardization (ISO) 14593: Carbon dioxide (CO2) Headspace Biodegradation
Test and provides a test for assessing the inherent aerobic biodegradability of
organic substances. It is particularly useful for testing insoluble and/or volatile
materials, and was successfully ring tested in 1996/97 using a formulated
hydraulic fluid, hexadecane, di-isotridecyl adipate and two mineral base oils.
(b) Purpose. (1) This guideline describes a screening method for the
evaluation of inherent biodegradability of chemical substances. The test is similar
to that described in OECD 310, Ready Biodegradability—CO2 in Sealed Vessels
(Headspace Test) (see paragraph (g)(2) of this guideline), in that both are based
on measurement of CO2 as inorganic carbon in either the headspace or liquid
phase. However, this test uses a mildly pre-exposed inoculum and thus meets the
definition of a test for inherent biodegradability. Unlike other inherent
biodegradation tests, most notably Zahn-Wellens/EMPA (OECD 302B) (see
paragraph (g)(3) of this guideline) and the Semi-Continuous Activated Sludge test
(OECD 302A) (see paragraph (g)(4) of this guideline), this method can be used to
test insoluble substances, since biodegradation measurements are based on CO2
production rather than dissolved organic carbon (DOC) disappearance. The
similar US Environmental Protection Agency Test Guideline 835.3100 (see
paragraph (g)(5) of this guideline) also uses a pre-exposed inoculum and
measures CO2 production, but does not include the improvements in measurement
of inorganic carbon that are in OECD 310 (see paragraph (g)(2) of this guideline)
and which solved difficulties experienced with the Sturm test (OECD 301B) and
similar methods (see paragraph (g)(2) of this guideline).
(2) A substance yielding a positive result in this test may be classified as
inherently biodegradable, which, preferably, should be qualified by the term "with
pre-adaptation". Because of the favorable conditions employed in this test, rapid
biodegradation in the environment of inherently biodegradable chemicals cannot
be assumed.
(c) General considerations—(1) Principle of the test, (i) The test
substance is incubated in a buffered, mineral salts medium which has been
inoculated with a mixed population of microorganisms. In order to enhance the
biodegradative potential of the inoculum, it is pre-exposed to the test substance
using a regime based on methods described in US Environmental Protection
Agency Test Guideline 835.3100 (see paragraph (g)(5) of this guideline). The test
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is performed in sealed bottles with a headspace of air that provides a reservoir of
oxygen (02) for aerobic biodegradation. CC>2 evolution from the ultimate aerobic
biodegradation of the test substance is determined by measuring the inorganic
carbon (1C) produced in the test bottles over that produced in blanks which
contain inoculated medium only. Aerobic ultimate biodegradation is the
breakdown of an organic chemical by microorganisms in the presence of C>2,
resulting in the production of CC>2, water, mineral salts of various elements and
microbial cellular constituents (biomass). The extent of biodegradation is then
expressed as a percentage of the theoretical maximum 1C production (ThIC),
based on the quantity of test substance (as total organic carbon) added initially.
(ii) DOC removal (water-soluble substances only) and/or the extent of
primary biodegradation of the test substance can also be measured. Primary
biodegradation is the structural change (transformation) of an organic chemical by
microorganisms resulting in the loss of a specific property.
(2) Information on the test substance. The organic carbon content (%
w/w) of the test substance needs to be known either from its chemical structure or
by measurement. For volatile test substances, a measured or calculated Henry's
law constant is helpful for determining a suitable headspace-to-liquid ratio.
Information on the toxicity of the test substance to bacteria is useful for selecting
an appropriate test concentration and for interpreting results showing poor
biodegradability. As this test is usually performed only after failure to pass a test
for ready biodegradability (see paragraph (g)(6) and (g)(7) of this guideline), the
physical and inhibitory properties may already have been ascertained.
(3) Applicability of the method. The test is applicable to water-soluble
and insoluble test substances. Using the recommended headspace to liquid ratio of
1:2, volatile substances with a Henry's law constant of up to 50 Pa m3 mol"1 can
be tested, as the proportion of test substance in the headspace will not exceed 1%
(see paragraph (g)(8) of this guideline). A smaller headspace volume may be used
when testing more volatile substances. However, users should ensure that the
headspace-to-liquid ratio and the test substance concentration are such that
sufficient C>2 is available to allow complete aerobic biodegradation to occur (e.g.,
avoid using both a high substrate concentration and a small headspace volume).
Guidance on this can be found in paragraph (g)(9) of this guideline.
(4) Reference substances (i) In order to check the test procedure, a
reference substance of known biodegradability should be tested in parallel. For
this purpose, w-hexadecane or a rapeseed (Canola) oil with a low (<2% w/w)
erucic acid content, is recommended when testing insoluble test substances.
Sodium benzoate is recommended when testing water-soluble test substances.
Biodegradation of these substances should reach 60% ThIC by the end of the test.
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(ii) To demonstrate the increased biodegradative power of the test over a
ready biodegradability test, di-isotridecyl adipate (DITA) can be used as a more
difficult-to-biodegrade reference substance. DITA is typically biodegraded by
only about 30% ThIC after 28 days with an unexposed inoculum (e.g. in a 301B
test; see paragraph (g)(6) of this guideline), but can be mineralized by 40-80%
ThIC in this test. DITA is a reference oil (RL 130) for the CEC L-33-A-93
biodegradability test and details on how to obtain it can be found in the reference
in paragraph (g)(10) of this guideline.
(5) Reproducibility—(i) Based on the CONCAWE 1996/97 ring test of
the method (see paragraph (g)(l) of this guideline), the difference between two
single and independent test results obtained by different operators, working in
different laboratories and on the same test substance, would exceed 38% only in
one case in 20.
(ii) In the CONCAWE 1996/97 ring test of the method (see paragraph
(g)(l) of this guideline), the results in Table 1 were obtained using the
recommended test conditions.
Table 1. Results from 1996/97 CONCAWE ring test for two substances.
Test substance
(Both insoluble)
w-Hexadecane
Di-isotridecyl adipate (DITA)
Mean Percentage
biodegradation
(Day 56)
72
65
Coefficient of
variation
(%)
21%
21%
Number of
laboratories
10
10
For these two insoluble test substances, the variability (as the coefficient of
variation) between replicates in the same test run was <10% for laboratories that
were experienced in using the method.
(d) Description of the method—(1) Apparatus. Normal laboratory
apparatus and the equipment in paragraphs (d)(l)(i) through (d)(l)(ix) of this
guideline.
(i) pH meter;
(ii) Analytical instruments such as for gas-liquid, high-performance liquid
or thin layer chromatography;
(iii) Glass serum bottles, sealed with butyl rubber stoppers and crimp-on
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aluminum seals. The recommended nominal size is 125 ml, which has an actual
total volume ca. 160 ml;
(iv) Carbon analyzer or other instrument(s) (e.g. gas chromatograph) for
measuring inorganic carbon;
(v) Syringes of high precision for gaseous and liquid samples;
(vi) Orbital shaker in a temperature-controlled environment;
(vii) A supply of CO2-free air. This can be prepared by passing air through
soda lime granules or by using an 80% N2/20% O2 gas mixture;
(viii) Membrane filtration device of 0.20 - 0.45 jim porosity (optional);
(ix) Organic carbon analyzer (optional).
(2) Reagents. Use analytical grade reagents throughout.
(3) Water. Distilled or deionized water that contains less than or equal to
1 mg total C per L should be used. This amount of C represents at most 5% of the
initial organic carbon content introduced by the recommended dose of the test
substance.
(4) Stock solutions for the mineral salts medium (i) The stock solutions
and the mineral salts medium are similar to those employed in ISO 14593 (see
paragraph (a)(2) of this guideline) and Organization for Economic Cooperation
and Development (OECD) 301 ready biodegradability tests (see paragraphs (g)(6)
and (g)(7) of this guideline). However, it should be noted that solution A (see
paragraph (d)(4)(iii)(A) of this guideline) contains a higher concentration of
ammonium chloride to reduce the possibility of biodegradation becoming
nitrogen-limited.
(ii) Stock solutions should be stored under refrigeration and discarded
after six months, or earlier, if there is evidence of microbial growth or
precipitation.
(iii) Prepare the stock solutions in paragraphs (d)(4)(iii)(A) through
(A) Solution A.
Potassium dihydrogen phosphate (KH^PO^ ....................................... 8.50g
Dipotassium hydrogen phosphate (K^HPO^ 21.75g
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Disodium hydrogen phosphate dihydrate (Na2HPO4-2H2O) 33.40g
Ammonium chloride (MLjCl) 2.00g
Dissolve in water and add water to 1 liter. The pH of this solution should
be 7.4 (± 0.2). If this is not the case, prepare a new solution.
(B) Solution B.
Calcium chloride dihydrate (CaCl2-2H2O) 36.40g
Dissolve in water and add water to 1 liter.
(C) Solution C.
Magnesium sulfate heptahydrate (MgSO4.7H2O) 22.50g
Dissolve in water and add water to 1 liter.
(D) Solution D.
Iron (III) chloride hexahydrate (FeCl3.6H20) 0.25g
Dissolve in water and add water to 1 liter, then add one drop of
concentrated HC1.
(5) Preparation of the mineral salts medium. Mix 10 ml of solution A
with approximately 800 ml water (see paragraph (d)(3) of this guideline), then
add 1 ml each of solutions B, C and D, and add water to 1 liter.
(6) Inoculum, (i) General. The test uses a composite microbial inoculum,
derived from soil and a wastewater treatment plant, which has been preexposed to
the test substance. In order to maximize the biodegradation potential of the test,
samples can be taken from locations where preexposure to the test substance or
similar materials may already have occurred (e.g. contaminated soil, industrial
biotreater).
(ii) Soil. Clear the soil surface of leaf litter and collect a sample to a depth
of up to 20 cm below the soil surface. Transport in a loosely tied black
polyethylene bag. Remove stones, plant debris and invertebrates from the soil and
pass through a 2 mm mesh sieve (if the soil is too wet to sieve immediately, then
partially air dry the soil to facilitate sieving). If the sieved soil will not be used
immediately, then it can be stored at 2 to 4°C in a loosely-tied black polythene
bag for up to one month. Further guidance on collection and handling of soil
samples may be found in the reference in paragraph (g)(l 1) of this guideline.
(iii) Activated sludge. Collect activated sludge from a wastewater
treatment plant and keep aerated at 20 ± 1°C until use (this should be within two
days of collection).
(iv) Preexposure procedure. (A) To 1 liter mineral salts medium, add 1 g
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prepared soil, 2 ml activated sludge and 50 mg/L yeast extract (e.g. Oxoid, Difco).
Adjust the pH (if necessary) to 7.4 ± 0.2 and dispense 100 ml aliquots into conical
flasks of 250 to 300 ml volume. Keep the suspension well mixed while
dispensing. Add 4 mg/L as carbon of test substance to replicate flasks. The
number of flasks needed will depend on the volume of inoculum needed to
perform the test. Close the flasks with stoppers that will allow gaseous exchange
(e.g. foam stoppers or aluminum caps) and incubate at 20 ± 1°C, with shaking or
mixing at a rate sufficient to keep the flask contents well mixed and in
suspension. Losses of a volatile test substance may occur during this procedure.
(B) On or around day 7 and day 11 of incubation, make up any
evaporation losses of test medium with water, adjust the pH (if necessary) to
between 7.2 and 7.6, and add 8 mg/L as carbon of test substance to each flask.
The final addition of test substance should be made at least three days before the
inoculum is used for the test.
(C) On the day of the test (normally day 14 of preexposure) coarse-filter
the inoculum through a number 4 grade filter paper or glass wool and keep shaken
until use. If two or three substances are being tested, the preexposed cultures for
each test substance can be pooled to give a composite inoculum. This reduces the
number of blanks needed and provides a microbial population that has been
exposed to more than one test substance.
(D) The inoculum is normally used within one day. If problems with
invalid (i.e. too high) blank inorganic carbon production (see paragraph (f)(4)(i)
of this guideline) have been encountered, or are thought likely to occur, then one
or both of the following treatments designed to reduce inorganic carbon content
are recommended:
Treatment 1: Aerate the inoculum overnight before use;
Treatment 2: Before use, sparge the inoculum with CO2-free air for about
one hour, while maintaining the pH at 6.5 using orthophosphoric acid. Finally,
restore the pH to its original value.
(e) Test procedure—(1) Number of bottles, (i) The number of bottles
needed for a test will depend on the frequency of analysis and the test duration. It
should be noted that the test may have to be continued beyond the 56 days
suggested in paragraph (e)(5) of this guideline.
(ii) It is recommended that at least five bottles from each of bottle sets 1, 2
and 3 (see paragraph (e)(3)(ii) of this guideline) be analyzed at the end of the test,
to enable 95% confidence intervals to be calculated for the mean percentage
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biodegradation value.
(2) Inoculated medium. The inoculum is used at a concentration of 10%
v/v (e.g. add 100 ml inoculum to 900 ml mineral salts medium).
(3) Preparation of bottles, (i) Aliquots of inoculated medium are
dispensed into replicate bottles to give a headspace-to-liquid ratio of 1:2 (e.g. add
107 ml to 160 mi-capacity bottles). Other ratios may be used but see the warning
given in paragraph (c)(3) of this guideline.
(ii) Sets of bottles should be prepared as outlined in paragraphs
(e)(3)(ii)(A) through (e)(3)(ii)(E) of this guideline.
(A) Bottle set 1: Contains test substance (held on a glass fiber filter, if
necessary-see paragraph (e)(3)(iv) of this guideline) at a recommended
concentration of 20 mg/L as organic carbon;
(B) Bottle set 2: Blanks containing inoculated medium (and a glass fiber
filter, if necessary) only;
(C) Bottle set 3: Contains reference substance at a recommended
concentration of 20 mg/L as organic carbon (and a glass fiber filter, if necessary).
(D) Bottle set 4: Same as bottle set 1, plus 50 mg/L HgCl2 or sterilized by
some other means (e.g. by autoclaving).
(E) Bottle set 5: Contains the test substance plus the reference substance
at the same concentrations used in bottle sets 1 and 3.
(iii) Water-soluble test and reference substances are added from aqueous
stock solutions.
(iv) Insoluble test and reference substances can be added to bottles as a
measured weight absorbed on a GF/A glass fiber filter (21 mm diameter) or by
direct injection using a microsyringe.
(A) Use of a glass fiber filter enables an accurate weight to be dosed into
each bottle and increases bioavailability. Weighing can be facilitated by preparing
a stock solution of the substance in a suitable volatile solvent (e.g.
dichloromethane) and adding the required volume dropwise to a tared filter on an
analytical balance. An attempt should be made to spread the substance over the
filter. The solvent is then allowed to evaporate until a stable weight (or nominal
test weight) is obtained. For example, 100 ^1 of a 21.4 mg/mL as carbon stock
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solution of test substance added to a filter gives 2.14 mg carbon as test
substance/filter, thus 20 mg carbon/L when added to 107 ml of inoculated
medium. The measured weight of test substance on the filter should be used to
calculate the test dose (see paragraph (f)(l)(i) of this guideline).
(B) If insoluble test or reference substance is injected into sealed bottles
using a microsyringe, the syringe should be weighed before and after dosing to
enable an accurate test dose to be calculated.
(v) If the procedure given in paragraph (e)(3)(iv)(A) of this guideline is
used, then blank bottles (bottle set 2) should contain either a filter or a filter from
which an equivalent volume of solvent has been evaporated (removal of the
solvent should be checked by weighing).
(vi) Volatile test substances should be injected into sealed bottles using a
microsyringe. The syringe should be weighed before and after dosing to enable an
accurate test dose to be calculated.
(vii) All the bottles are then sealed and time zero analyses are performed
for initial 1C concentration, DOC concentration, or other determinants.
(4) Incubation. The bottles are incubated in the dark or diffuse light at 20
± 1°C, with shaking at a rate sufficient to keep the bottle contents well mixed and
in suspension (e.g. 150 to 200 rpm).
(5) Sampling. Replicate test (Bottle set 1), blank (Bottle set 2), reference
(Bottle set 3), and inhibition control (Bottle set 5) bottles should be taken for 1C
analysis periodically, in order to obtain the biodegradation curves. Normally this
is done at least once each week. At the end of the test, the sterile controls (Bottle
set 4) should also be analyzed. The test normally runs until biodegradation
reaches a plateau. As a guide, users should plan for an eight-week test. However,
the test can be continued if biodegradation is still occurring on day 56. It is not
advisable to continue the test beyond three months.
(6) Inorganic carbon (1C) analysis, (i) CC>2 production in the bottles is
determined by measuring the increase in the concentration of 1C. There are two
methods available for measuring the amount of 1C produced in the test, described
in paragraphs (e)(7) and (e)(8) of this guideline. As the methods can give slightly
different results, only one should be used in a given test run.
(ii) Method A (see paragraph (e)(7) of this guideline) is recommended if
the liquid medium is likely to contain the remnants of a GF/A filter and/or
insoluble test substance. This analysis can be performed using a gas
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chromatograph if a carbon analyzer is unavailable. It is important that the bottles
should be at or close to the test temperature of 20 ± 1°C when the headspace gas
is analyzed. Method B (see paragraph (e)(8) of this guideline) can be easier for
laboratories using carbon analyzers to measure 1C. It is important that the sodium
hydroxide solution used to convert CC>2 to carbonate is either freshly prepared or
its 1C content is known, so that this can taken into account when calculating the
test result.
(7) Method A: acidification to pH <3. (i) Calibrate the 1C analyzer using
an appropriate 1C standard (e.g. 1% w/w CC>2 in N2). Inject concentrated (85%
w/v) orthophosphoric acid through the septum of each test bottle sampled to lower
the pH of the medium to <3 (e.g. add 1 ml to 107 ml test medium). Replace the
bottles on the shaker. After shaking for one hour at the test temperature (20 ±
1°C), remove the bottles from the shaker, withdraw aliquots (e.g. 1 ml) of gas
from the headspace of each bottle and inject into the 1C analyzer. Record the
measured 1C concentration (mg carbon/L).
(ii) The principle of this method is that after acidification to pH <3 and
equilibration at 20°C, the equilibrium constant for the distribution of CCh between
the liquid and gaseous phases in the test bottles is 1.0 (see paragraphs (g)(l),
(g)(8) and (g)(12) of this guideline). This should be demonstrated for the test
system at least once (e.g. at the end of the test) as follows: set up bottles
containing 5 and 10 mg IC/L using a solution of anhydrous sodium carbonate
(Na2CO3) in CO2-free water (prepared by acidifying water to pH 6.5 with
concentrated orthophosphoric acid, sparging overnight with CCh-free air and
raising the pH to neutrality with alkali). Ensure that the ratio of the headspace
volume to the liquid volume is the same as in the tests (e.g. 1:2). Acidify and
equilibrate as described in paragraph (e)(7)(i) of this guideline, and measure the
1C concentrations of both the headspace and liquid phases. Check that the two
concentrations are the same within experimental error.
(iii) If DOC removal is to be measured (water-soluble test substances
only), take samples of the liquid phase from separate (unacidified) bottles,
membrane filter and inject into the carbon analyzer. These bottles can be used for
other analyses as necessary.
(8) Method B: conversion of COi to carbonate, (i) Calibrate the 1C
analyzer using an appropriate standard-for example, a solution of sodium
bicarbonate (NaHCOs) in CO2-free water in the range 0 to 20 mg IC/L. Inject 7 M
sodium hydroxide (e.g. 1 ml to 107 ml of medium) through the septum of each
test bottle sampled and shake for one hour at the test temperature (20 ± 1°C).
Remove the bottles from the shaker, allow to settle, and withdraw, by syringe,
suitable volumes (e.g. 50 to 200 jil) from the liquid phase in each vessel. Inject
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the samples into the 1C analyzer and record the concentration of 1C.
(ii) The principle of this method is that after the addition of alkali and
shaking, the concentration of 1C in the headspace is negligible. This should be
checked for the test system at least once (e.g. at the end of the test) by using 1C
standards, adding alkali and equilibrating, and measuring the concentration of 1C in
both the headspace and liquid phases.
(iii) If DOC removal is to be measured (water-soluble test substances
only), take samples of the liquid phase from separate bottles (no alkali),
membrane filter and inject into the carbon analyzer. These bottles can be used for
other analyses as necessary.
(f) Data and reporting—(1) Calculation of results, (i) Assuming 100%
mineralization of the test substance to CC>2, the theoretical maximum 1C
production (ThIC) in excess of that produced in the blanks equals the amount of
total organic carbon (TOC) from the test substance added to each bottle at the
start of the test. For insoluble substances, TOC should be calculated from the
measured weight of test substance (absorbed onto the filter or added by
microsyringe), and its organic carbon content. For volatile substances, TOC
should be calculated from the measured weight of test substance dispensed by
microsyringe, and its organic carbon content.
The total mass (mg) of inorganic carbon (TIC) in each bottle is:
TIC = (mg C in the liquid + mg C in the headspace)
= (VL x CL) + (VH x CH) Equation [ 1 ]
where :
VL = volume of liquid in the bottle (liter);
CL = concentration of 1C in the liquid (mg/L as carbon);
VH = volume of the headspace (liter);
CH = concentration of 1C in the headspace (mg/L as carbon).
(ii) The calculation of TIC for the two analytical methods used for
measuring 1C in this test is described in this paragraph. Percentage biodegradation
(% D) in each case is given by:
%D = (TICt-TICh) x 100
TOC
where :
TICt = mg TIC in test bottle at time t;
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TICb = mean mg TIC in blank bottles at time t;
TOC = mg TOC added initially to the test vessel.
Determine total %D for the test (Bottle set 1), reference (Bottle set 3) and
inhibition control bottles (Bottle set 5), from the cumulative amounts of TIC
produced up to each sampling time.
(iii) If there has been a significant increase in the TIC content of the sterile
controls (Bottle set 4) over the test period, then abiotic degradation of the test
substance has occurred.
(2) Calculation of TIC for analytical Method A. Acidification to pH <3
and equilibration results in the equalization of the concentration of 1C in the liquid
and gaseous phases. Hence only the concentration of 1C in the gas phase needs to
be measured, as CL = CH. Therefore TIC = (VL + VH) x CH in Equation [1].
(3) Calculation of TIC for analytical Method B. In this method
calculations are performed as described in Equation [1] but the negligible amount
of 1C in the gaseous phase is ignored; i.e. VH x CH in Equation [1] is set to zero.
(4) Validity of results, (i) The test is considered as valid if:
(A) The mean percentage biodegradation of n -hexadecane, low erucic
acid-containing rapeseed oil (canola oil) or sodium benzoate reaches at least 60%
ThIC by the end of the test; and
(B) The mean amount of 1C produced from the blanks at the end of the test
is no more than 15% of the organic carbon added initially as the test substance to
the test bottles.
(C) If these criteria are not met, repeat the test with another inoculum
and/or review procedures. For example, if high blank 1C production is a problem
then follow the procedures given in paragraph (d)(6)(iv)(D) of this guideline.
(ii) It can be assumed that the test substance is inhibitory if the mean
percentage degradation in the inhibition control bottles (Bottle set 5) is <25% by
the end of the test. In this case, the test can be repeated with a lower concentration
of test substance (e.g. 10 mg/L as carbon). However, it should be noted that this
will reduce the precision of the method. If lower test substance concentrations are
used, it is important that blank 1C production is as low as possible and that the
smaller amount of test substance can be dosed accurately.
(5) Interpretation of results, (i) Biodegradation greater than 70% ThIC
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in this test demonstrates that the test substance is inherently and ultimately
biodegradable under aerobic conditions.
(ii) Biodegradation less than or equal to 20% ThIC is an indication that the
test substance is not inherently biodegradable under the conditions of this test.
However, this does not rule out metabolism under different circumstances (e.g.
with a longer preexposure period and/or higher biomass-to-test substance ratio).
(iii) Biodegradation greater than 20% ThIC is an indication that the test
substance has inherent, primary biodegradability.
(iv) If more information is needed on the potential fate of the test
substance, then further investigations such as DOC analysis to detect water-
soluble metabolites may be useful.
(6) Test report, (i) Compile a table of %D for each test (Bottle set 1),
reference (Bottle set 3) and inhibition control bottle (Bottle set 5) for each day
sampled. If comparable results are obtained for replicate bottles, plot a curve of
mean %D against time. Record the amount of TIC in the blanks (Bottle set 2), and
in the sterile controls (Bottle set 4). If determined, also record the levels of DOC
and/or other determinants and their percentage removal.
(ii) Determine the mean value of %D in the plateau phase, or use the
highest value if the biodegradation curve decreases after peaking, and report this
as the extent of biodegradation of the test substance. It is important to ensure that
in the latter case the highest value is not an outlier.
(iii) The test report should include the information in paragraphs
(f)(6)(iii)(A) through (f)(6)(iii)(C) of this guideline:
(A) Test substance:
(1) Common name, chemical name, CAS number, structural formula and
relevant physical-chemical properties;
(2) Purity (impurities) of test substance.
(B) Test conditions:
(1) Description of the test system used (e.g. volume of the vessel,
headspace-to-liquid ratio, method of stirring, etc);
(2) Application of test substance and reference substance to test system:
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test concentration used and amount of carbon dosed into each test bottle, and any
use of solvents;
(3) Details of the inoculum used, its preexposure to the test substance and
any preconditioning (e.g. overnight aeration);
(4) Incubation temperature;
(5) Validation of the principle of 1C analysis;
(6) Main characteristics of the 1C analyzer employed (and any other
analytical methods used);
(7) Number of replicates.
(C) Results:
(1) Raw data and calculated values of biodegradability in tabular form and
degradation curves;
(2) Reasons for any rejection of the test results;
(3) Any other facts that are relevant to the procedure followed;
(4) Discussion of results.
(g) References.
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