United States Prevention, Pesticides EPA712-C-08-001
Environmental Protection And Toxic Substances October 2008
Agency (7101)
4>EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3140
Ready Biodegradability
—CO2 in Sealed Vessels
(Headspace 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 835.3140: Ready biodegradability—CO2 in sealed vessels
(Headspace 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 OPPTS test guideline is based on the OECD
Guideline for the Testing of Chemicals no. 310, Ready Biodegradability - CO2 in
Sealed Vessels (Headspace Test) (adopted 23 March 2006), available from source
OECD at http://masetto.sourceoecd.or:g and U.S. EPA Fate, Transport and
Transformation Test Guidelines no. 835.3120, Sealed-Vessel CO2 Production
Test (Jan. 1998), available from http://www.epa.gov/opptsfrs/home/guidelin.htm.
(b) Purpose. This guideline is a screening method for the evaluation of
ready biodegradability of chemical substances and provides similar information to
that from the six test methods described in OECD Test Guideline 301 A to F (see
paragraph (o)(l) of this guideline). Therefore, a chemical substance that shows
positive results in this guideline can be considered readily biodegradable and
consequently rapidly degradable in the environment.
(c) General considerations. (1) The well-established OECD carbon
dioxide (CO2) method (i.e. 301B) (see paragraph (o)(l) of this guideline), based
on Sturm's original test (see paragraph (o)(2) of this guideline) for assessing
biodegradability of organic chemicals, by the measurement of the CO2 produced
by microbial action, has normally been the first choice for testing poorly soluble
chemicals and those that strongly adsorb. It is also chosen for soluble (but not
volatile) chemicals, since the evolution of CO2 is considered by many to be the
only unequivocal proof of microbial activity. Removal of DOC can be effected by
physical or chemical processes - adsorption, volatilization, precipitation, and
hydrolysis - as well as by microbial action, and many nonbiological reactions
consume oxygen. However, rarely is CO2 produced from organic chemicals
abiotically. In the original and modified Sturm test, CO2 is removed from the
liquid phase to the absorbing vessels by sparging (i.e. bubbling air treated to
remove CO2 through the liquid medium), while in the version of Larson (see
paragraphs (o)(3) and (o)(4) of this guideline), CO2 is transferred from the
reaction vessel to the absorbers by passing CO2-free air through the headspace
and, additionally, by shaking the test vessel continuously. Only in the Larson
modification is the reaction vessel shaken; stirring is specified only for insoluble
substances in the ISO version (see paragraph (o)(5) of this guideline) and in the
original US version (see paragraph (o)(6) of this guideline), both of which specify
sparging rather than headspace replacement. In another official US EPA method
(see paragraph (o)(7)) of this guideline based on Gledhill's method (see paragraph
(o)(8) of this guideline), the shaken reaction vessel is closed to the atmosphere and
CO2 produced is collected in an internal alkaline trap directly from the gaseous
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phase, as in classical Warburg/Barcroft respirometer flasks.
(2) However, 1C has been shown to accumulate in the medium during the
application of the modified Sturm test (see paragraph (o)(9) of this guideline). A
concentration of 1C as high as 8 mg/L was found during the degradation of 20 mg
C/L of aniline. Thus, the collection of CO2 in the alkaline traps did not give a true
reflection of the amount of CO2 produced microbiologically at intermediate times
during the degradation. As a result, the specification that >60% of ThCO2 should
be collected within a 10-d window for a test substance to be classified as readily
biodegraded will not be met for some substances that would be so classified using
DOC removal.
(3) When the percentage degradation is a lower value than expected, 1C
has possibly accumulated in the test solution. Then, the degradability may be
assessed with the other OECD ready biodegradability tests.
(4) Other drawbacks of the Sturm methodology (cumbersome test flasks;
time-consuming; more prone to experimental error; applicable to volatile
substances) had earlier prompted a search for a sealed vessel technique, other than
Gledhill's, rather than one based on gas flow-through (see paragraphs (o)(10) and
(o)(ll) of this guideline). Boatman et al. (see paragraph (o)(12) of this guideline)
reviewed the earlier methods and adopted an enclosed headspace system in which
the CO2 was released into the headspace at the end of incubation by acidifying the
medium. CO2 was measured by gas chromatography (GC)/IC analysis in
automatically taken samples of the headspace, but DIG in the liquid phase was not
taken into account. Also, the vessels used were very small (20 ml), containing
only 10 ml of medium, which caused problems e.g. when adding the necessarily
very small amounts of insoluble test substances. Another shortcoming is that due
to the amount of inoculated test medium, there may in some cases be insufficient
or no microorganisms present in the inoculated medium that are competent to
degrade a given test substance.
(5) These difficulties have been overcome as a results of the independent
studies of Struijs and Stoltenkamp (see paragraph (o)(13)) and of Birch and
Fletcher (see paragraph (o)(14) of this guideline). In the former study CO2 was
measured in the headspace after acidification and equilibration, while in the latter,
DIG in both the gaseous and liquid phases was measured, without treatment; over
90% of the 1C formed was present in the liquid phase. Both methods have
advantages over the Sturm test in that the test system is more compact and
manageable, volatile chemicals can be tested, and the possibility of delay in
measuring CO2 produced is avoided.
(6) The two approaches were combined in the ISO Headspace CO2
Standard (see paragraph (o)(15) of this guideline), which was ring-tested (see
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paragraph (o)(16) of this guideline) and it is this standard that forms the basis of
the present guideline. Two methods of measuring CC>2 have been recommended;
namely, CC>2 in headspace after acidification, and 1C in the liquid phase after the
addition of excess alkali. The latter method was introduced during the
CONCAWE ring test of this headspace method modified to measure inherent
biodegradability (see paragraph (n)(17) of this guideline). The changes made in
the 1992 revision of OECD Guidelines for Ready Biodegradability have been
incorporated into this guideline (see paragraph (o)(ll) of this guideline), so that
the conditions (medium, duration, etc.) are otherwise the same as those in the
revised Sturm test. Birch and Fletcher (see paragraph (o)(14) of this guideline)
obtained very similar results with this headspace test as were obtained with the
same chemicals in the 1988 OECD ring test of the revised ready biodegradability
test methods (see paragraph (o)(18) of this guideline).
(d) Definitions.
DIC: Dissolved inorganic carbon
DOC: Dissolved organic carbon is the organic carbon present in solution
or that which passes through a 0.45 micrometer filter or remains in the
supernatant after centrifuging at approx. 4000 g (about 40.000 m sec"2) for 15
min.
1C: Inorganic carbon
Inherent Biodegradability: A classification of chemicals for which there is
unequivocal evidence of biodegradation (primary or ultimate) in any test of
biodegradability.
Lag Phase: The time from the start of a test until acclimatization and/or
adaptation of the degrading microorganisms is achieved and the biodegradation
degree of a chemical substance or organic matter has increased to a detectable
level (e.g. 10% of the maximum theoretical biodegradation, or lower, dependent
on the accuracy of the measuring technique).
Mineralization: Mineralization is the complete degradation of an organic
compound to CC>2 and H2O under aerobic conditions, and CH4, CC>2 and H2O
under anaerobic conditions.
Plateau phase: Plateau phase is the phase in which the maximal
degradation has been reached and the biodegradation curve has leveled out.
Readily Biodegradable: An arbitrary classification of chemicals which
have passed certain specified screening tests for ultimate biodegradability; these
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tests are so stringent that it is assumed that such substances will rapidly and
completely biodegrade in aquatic environments under aerobic conditions.
ThCO2: Theoretical carbon dioxide (mg) is the quantity of carbon dioxide
calculated to be produced from the known or measured carbon content of the test
substance when fully mineralized; also expressed as mg carbon dioxide evolved
per mg test substance.
ThIC: Theoretical inorganic carbon
TIC'. Total inorganic carbon
Ultimate Aerobic Biodegradation: The level of degradation achieved when
the test substance is totally utilized by micro-organisms resulting in the
production of carbon dioxide, water, mineral salts and new microbial cellular
constituents (biomass).
10-d window: The 10 days immediately following the attainment of 10%
biodegradation.
(e) Principle of the test. (1) The test substance, normally at 20 mg C/L, as
the sole source of carbon and energy, is incubated in a buffer-mineral salts
medium that has been inoculated with a mixed population of microorganisms
from sewage treatment effluent obtained from a local domestic sewage treatment
plant. The test is performed using sealed bottles with a headspace of air, which
provides a reservoir of oxygen for aerobic biodegradation. The CC>2 evolution
resulting from the ultimate aerobic biodegradation of the test substance is
determined by measuring the 1C produced in the test bottles in excess of that
produced in blank vessels containing inoculated medium only. The extent of
biodegradation is expressed as a percentage of the theoretical maximum 1C
production (ThIC), based on the quantity of test substance (as organic carbon)
added initially.
(2) The DOC removal and/or the extent of primary biodegradation of the
test substance can also be measured for water soluble materials that do not adsorb
to glass or biological solids.
(f) Applicable ASTM standards. Refer to the documents referenced in
paragraph (o)(19) of this guideline for the standards in paragraphs (f)(l) through
(f)(8) of this guideline:
(1) Dl 129-90: Standard Terminology Relating to Water.
(2) Dl 193-91: Standard Specifications for Reagent Water (Federal Test
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Method and Standard No. 7916).
(3) D1293-84: Standard Test Methods for pH of Water.
(4)D2579-85: Standard Test Method for Total and Organic Carbon in
Water.
(5) D2777: Standard Practice for Determination of Precision and Bias of
Applicable Methods of Committee D-19 on Water.
(6) D4375-90: Standard Terminology for Basic Statistics in Committee
D-19 on Water.
(7) D4839-88: Standard Test Method for Total Organic Carbon in Water
by Ultraviolet, or Persulfate Oxidation or Both, and Infrared Detection.
(8) E178-80: Standard Practice for Dealing with Outlying Observations.
(g) Information on the test substance. The organic carbon content (%
w/w) of the test substance should be known, either from its chemical structure or
by measurement, so that the percentage degradation may be calculated. For
volatile test substances, a measured or calculated Henry's law constant is helpful
for determining a suitable headspace-to-liquid volume ratio. Information on the
toxicity of the test substance to microorganisms such as from an Activated Sludge
Respiration Inhibition test (OECD 209; see paragraph (o)(20) of this guideline), is
useful in selecting an appropriate test concentration and for interpreting results
showing poor biodegradability. It is also recommended to include the inhibition
control unless it is known that the test substance is not inhibitory to microbial
activities (see paragraph (1)(9) of this guideline).
(h) Applicability of the method. The test is applicable to insoluble as
well as water-soluble test substances, but good dispersion of the substance should
be ensured. Using the recommended headspace-to-liquid volume ratio of 1:2,
volatile substances with a Henry's law constant of up to 50 Pa.m3.mor1 can be
tested, as the proportion of test substance in the headspace will not exceed 1%
(see paragraph (o)(13) of this guideline). A smaller headspace volume may be
used when testing substances that are more volatile, but their bioavailability may
be limiting especially if they are poorly soluble in water. However, users should
ensure that the headspace-to-liquid volume ratio and the test substance
concentration are such that sufficient oxygen is available to allow complete
aerobic biodegradation to occur (e.g. avoid using a high test substance
concentration and a small headspace volume). Guidance on this can be found in
references cited in paragraphs (o)(13) and (o)(21) of this guideline.
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(i) Reference substances. In order to check the test procedure, a reference
substance of known biodegradability should be tested in parallel. For this purpose,
aniline, sodium benzoate or ethylene glycol may be used when testing water-
soluble test substances, and 1-octanol for poorly soluble test substances (see
paragraph (o)(13) of this guideline). Biodegradation of these substances must
reach >60% ThIC within 14 days.
(j) Reproducibility. (1) In the ISO ring test of the method (see paragraph
(o)(16) of this guideline), the results in Table 1 were obtained using the
recommended conditions, including 20 mg test substance as C/L.
Table 1. Summary of results from ISO ring test
(see paragraph (o)(16) of this guideline)
Test
Substance
Aniline
1-Octanol
Mean Percentage
Biodegradation
(28d)
90
85
Coefficient of variation
(%)
16
12
Number of
Laboratories
17
14
Within-test variability (replicability), using aniline, was low with coefficients of
variability not greater than 5% in nearly all test runs. In the two cases in which
the replicability was worse, the greater variability was probably due to high 1C
production in the blanks. Replicability was worse with 1-octanol, but still less
than 10% for 79% of test runs. This greater within-test variability may have been
due to dosing errors, as a small volume (3 to 4 jil) of 1-octanol had to be injected
into sealed test bottles. Higher coefficients of variation would result when lower
concentrations of test substance are used, especially at concentrations lower than
10 mg C/L. This could be partially overcome by reducing the concentration of
total inorganic carbon (TIC) in the inoculum.
(2) In an EU ring-test (see paragraph (o)(22) of this guideline) of five
surfactants added at 10 mg C/L, the results in Table 2 were obtained.
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Table 2. Summary of results from
(see paragraph (o)(22) of this c
Test Substance
Tetrapropylenebenzenesulphonate
Di-isooctylsulfosuccinate
Hexadecyltrimethylammonium
chloride
Isononylphenolethoxylate)
(NPE9)
Cocoamidopropyl
dimethylhydroxysulfobetaine
Mean Percentage
Biodegradation
(28d)
17
72
75
41
60
EU ring test
uideline)
Coefficient of
variation
(%)
45
22
13
32
23
Number of
laboratories
10
9
10
10
11
* SiO2was added to neutralize toxicity.
The results show that generally, the variability was higher for the less well-degraded
surfactants. Within-test variability was less than 15% for over 90% of cases, the highest reaching
30-40%.
NOTE: Most surfactants are not single molecular species but are mixtures of isomers,
homologues, etc. which degrade after different characteristic lag periods and at different kinetic
rates, resulting in blurred, extenuated curves, so that the 60% pass value may not be reached
within the 10-d window, even though each individual molecular species would reach >60% within
10 days if tested alone. This may be observed with other complex mixtures as well. See the
reference cited in paragraph (o)(23).of this guideline.
(k) Safety precautions. (1) This method involves the use of
nonchlorinated sewage treatment plant effluent. Consequently, individuals
performing this test may be exposed to microorganisms that are dangerous to
human health. Disposable latex gloves and laboratory eyewear with splash guards
should be worn during procedures involving the handling of effluent. When large
volumes of effluent are being handled, for example during filtering and sparging
operations, a dust/mist respirator and laboratory footwear should also be worn.
(2) Individuals who work with sewage microorganisms may want to keep
current with immunizations for polio, typhoid, hepatitis B, and tetanus.
(3) Test media should be treated with 5 percent chlorine bleach prior to
disposal.
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(1) Description of the method—(1) Apparatus, (i) Glass serum bottles
sealed with butyl rubber stoppers and crimp-on aluminum seals. The
recommended size is '125 ml' which have a total volume of approximately 160
ml (In this case the volume of each bottle should be known to be 160 ± 1 ml). A
smaller size of vessel may be used when the results fulfill the conditions
described in paragraph (n)(5) of this guideline);
(ii) Carbon analyzer or other instrument (e.g. gas chromatograph) for
measuring inorganic carbon. For analysis of carbon in the test medium (liquid
phase), the analyzer should be capable of measuring DIG and DOC in aqueous
media over the range of 0 to 20 mg C/L (a suitable instrument is an OI
Corporation model 700 TOC analyzer, or equivalent). For analysis of CO2 in
headspace gas, the analyzer should be capable of measuring CO2 over the range of
0 to 1 mg C (a suitable instrument is an Ionics model 1555b TOC analyzer with
Horiba model PIR2000 NDIR CO2 detector, or equivalent).
(iii) Syringes- A gas-tight, 1,000 mL cemented-needle syringe with 22°
beveled, bent point, for piercing butyl rubber or neoprene septa and injecting gas-
phase samples into the gas-phase analyzer (for example, Hamilton no. 1001 with
no. 81317 tip, or equivalent). A spring-loaded syringe with square end, for
injecting liquid samples into Ionics-type analyzers (or equivalent), if used.
(iv) Filter apparatus - Filter flask, 2- or 3-L capacity, 20-cm Biichner
funnel, 18.5-cm coarse filter paper (Whatman no. 41, or equivalent), and vacuum
source, for filtering sewage treatment effluent inoculum.
(v) Orbital shaker in a temperature-controlled environment;
(vi) 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 (optional);
(vii) Organic carbon analyzer (optional).
(viii) pH meter
(ix) Flasks/bottles - Three 100-mL volumetric flasks and one 1,000-mL
volumetric flask for preparing mineral nutrient medium stock solutions. One
2,000-mL volumetric flask for each test compound, for preparing test compound
stock solutions. For water-soluble test compounds that do not precipitate in the
presence of the mineral nutrient test medium, more concentrated stock solutions
may be used. In this case smaller volumetric flasks are suitable. Glass bottles or
flasks, 6-L capacity, for preparation of mineral nutrient medium.
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(x) Magnetic stirrers for media and sample preparation.
(xi) Automated pipetting device for delivering variable volumes of liquid
up to 100 mL with an accuracy of ± 1 percent (for example, EM Science Optifix,
or equivalent).
(xii) Large laboratory oven, for drying glassware after cleaning.
(xiii) Ultrasonic processor (sonicator), for dispersing sparingly soluble
test compounds, if needed.
(2) Reagents. Use analytical grade reagents throughout.
(3) Water, (i) Distilled or de-ionized water should be used containing <1
mg/L as total organic carbon. This represents <5% of the initial organic carbon
content introduced by the recommended dose of the test substance.
(ii) Deionized or distilled water should be free of calcium and toxic
substances, especially metals such as copper. It may be desirable to saturate the
water with oxygen before initiating the experiment by sparging for 20 min. with
clean, filtered compressed air.
(4) Preparation of mineral nutrient medium—(i) Stock solutions. The
stock solutions and the mineral salts medium are similar to those in ISO 14593
(see paragraph (o)(15) of this guideline) and OECD 301 ready biodegradability
tests (see paragraph (o)(l) of this guideline). The use of a higher concentration of
ammonium chloride (2.0 g/L instead of 0.5 g/L) should only be necessary in very
exceptional cases, e.g. when the test substance concentration is >40 mg C/L.
Stock solutions should be stored under refrigeration and disposed of after six
months, or earlier if there is evidence of precipitation or microbial growth.
Prepare the stock solutions in paragraphs (l)(4)(i)(A) through (l)(4)(i)(D) of this
guideline:
(A) Potassium dihydrogen phosphate (KH^PO/t) 8.50g
Dipotassium hydrogen phosphate (K^HPO/t) 21.75g
Disodium hydrogen phosphate dehydrate (Na2HPO4.2H2O) 33.40g
Ammonium chloride (NHtCl) O.SOg
Dissolve in water and make up to 1 liter. The pH of this solution should be
7.4 (± 0.2). If this is not the case, then prepare a new solution.
(B) Calcium chloride dihydrate (CaCl2.2H2O) 36.40g
Dissolve in water and make up to 1 liter.
(C) Magnesium sulfate heptahydrate (MgSO4.7H2O) 22.50g
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Dissolve in water and make up to 1 liter.
(D) Iron (III) chloride hexahydrate (FeCl3.6H20) 0.25g
Dissolve in water and make up to 1 liter and add one drop of concentrated
hydrolchloric acid.
(ii) Preparation of medium. Mix 10 ml of solution (A) with
approximately 800 ml water (see paragraph (1)(3) of this guideline), then add 1 ml
of solutions (B), (C) and (D) and add water to 1 liter.
(5) Other reagents, (i) Concentrated orthophosphoric acid (HaPO^
(>85% mass per volume).
(ii) CO2-free compressed air or nitrogen for sparging the inoculum to
ensure that it is free of dissolved CC>2. The delivery line should be equipped with
a large gas diffusing stone (Fisher no. 11-139A or equivalent) for maximum
sparging efficiency.
(iii) Calibration gas for headspace analysis should be of certified standard
grade and contain no more than approximately 0.25 percent CO2 by volume, with
the balance being nitrogen.
(6) Sodium hydroxide solution 7M. Dissolve 280 g of sodium hydroxide
(NaOH) in 1 liter of water (see paragraph (1)(3) of this guideline). Determine the
concentration of DIG of this solution and consider this value when calculating the
test result (see paragraphs (m)(7)(i) and (n)(3) of this guideline), especially in the
light of the validity criterion in paragraph (n)(5)(i)(B) of this guideline. Prepare a
fresh solution if the concentration of DIG is too high.
(7) Test substance, (i) Prepare a stock solution of a sufficiently water-
soluble test substance in water (see paragraph (1)(3) of this guideline) or in the test
medium (see paragraph (1)(4) of this guideline) at a concentration preferably 100-
fold greater than the final concentration to be used in the test. It may be necessary
to adjust the pH of the stock solution to 7.2 ± 0.2 with HC1 or NaOH. The stock
solution should be added to the mineral medium to give a final organic carbon
concentration of between 2 and 40 mg C/L, preferably 20 mg/C/L. If
concentrations lower than these are used, the precision obtained may be impaired.
Soluble and insoluble liquid substances may be added to the vessels directly using
high precision syringes. Test compounds known to be toxic to sludge
microorganisms at 10 mg C/L may be tested at lower initial concentrations down
to a minimum level of 2 to 5 mg C/L, the actual value depending on the
sensitivity of the carbon analyzer. Poorly soluble and insoluble test substances
may require special treatment (see paragraph (o)(24) of this guideline). The
choices are:
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(A) Direct addition of known weighed amounts;
(B) Ultrasonic dispersion before addition;
(C) Dispersion with the aid of emulsifying agents to be required to
establish whether they have any inhibitory or stimulatory effects on microbial
activity before addition;
(D) Adsorption of liquid test substances, or a solution in a suitable volatile
solvent, on to an inert medium or support (e.g. glass fiber filter), followed by
evaporation of the solvent, if used, and direct addition of known amounts;
(E) Addition of known volume of a solution of the test substance in an
easily volatile solvent to an empty test vessel, followed by evaporation of the
solvent.
(ii) Any solvents or dispersing agents should be tested for any stimulatory
or inhibitory effect on microbial activity (see paragraph (m)(3)(ii)(B) of this
guideline).
(8) Reference substance, (i) Prepare a stock solution of the (soluble)
reference substance in water (see paragraph (1)(3) of this guideline) at a
concentration preferably 100-fold greater than the final concentration to be used
(20 mg C/L) in the test.
(ii) Suitable substances are aniline (freshly distilled) and phthalic or
trimellitic acid, but sodium acetate, dextrose (glucose), and sodium benzoate may
be too biodegradable to be useful for this purpose.
(iii) For test compounds with very low or negligible water solubility, it
may be desirable to employ reference compounds of similar solubility; for
example, sodium stearate.
(9) Inhibition check. Test compounds frequently show no significant
degradation under the conditions used in ready biodegradation tests. One possible
cause is that the test substance is inhibitory to the inoculum at the concentration at
which it is applied. An inhibition check may be included in the test design to
facilitate identification (in retrospect) of inhibition as a possible cause or
contributory factor. Alternatively, the inhibition check may rule out such
interferences and show that zero or slight degradation is attributable solely to
nonsusceptibility to microbial attack under the conditions of the test. In order to
obtain information on the toxicity of the test substance to (aerobic) micro-
organisms, prepare a solution in the test medium containing both test substance
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and reference substance, each at the same concentration as when present
individually.
(10) Inoculum, (i) The inoculum may be derived from a variety of
sources: activated sludge; sewage effluent (non-chlorinated); surface waters and
soils; or a mixture of these (see paragraph (o)(l) of this guideline). The
biodegradative activity of the source should be checked by using a reference
substance. Whatever the source, microorganisms previously exposed to the test
substance should not be used if the procedure is to be used as a test for ready
biodegradability. See also the precautions in paragraph (k).
(ii) Based on experience, the optimal volume for the inoculum is that
which:
(A) Is sufficient to give adequate biodegradative activity;
(B) Degrades the reference substance by the stipulated percentage (see
paragraph (n)(5)(i) of this guideline);
(C) Gives 102 to 105 colony-forming units per milliliter in the final
mixture;
(D) Normally gives a concentration of 4 mg/L suspended solids in the
final mixture when activated sludge is used. Concentrations up to 30 mg/L may be
used but may significantly increase CC>2 production of the blanks (see paragraph
(o)(19) of this guideline);
(E) Contributes less than 10% of the initial concentration of organic
carbon introduced by the test substance;
(F) Is generally 1-10 ml of inoculum for 1 liter of test medium.
(iii) Inoculum may be preconditioned to the experimental conditions, in
order to reduce the blank CC>2 evolution. Preconditioning consists of aerating with
moist CO2-free air for up to 5-7 days at the test temperature, after diluting in test
medium, prior to starting the test.
(11) Activated sludge, (i) Activated sludge should be freshly collected on
the day of the experiment from the aeration tank of a sewage treatment plant
treating predominantly domestic sewage, or laboratory-scale unit treating
predominantly domestic sewage. If necessary, coarse particles should be removed
by sieving (e.g. using a 1 mm2 mesh sieve) and the sludge should be kept aerobic
until it is used.
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(ii) Alternatively, after removal of any coarse particles, settle or centrifuge
(e.g. 1100 x g for 10 minutes). Discard the supernatant liquid. The sludge may be
washed in the mineral solution. Suspend the concentrated sludge in mineral
medium to yield a concentration of 3-5 g suspended solids/L. Thereafter aerate
until it is used.
(iii) Sludge should be taken from a properly working conventional (i.e.
activated sludge process) 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 liquid, and again suspend the washed sludge in a further volume of
mineral medium. Repeat this procedure until the sludge is considered to be free
from excess substrate or inhibitors.
(iv) After complete resuspension is achieved, or with untreated sludge,
withdraw a sample just before use for the determination of the dry weight of the
suspended solids.
(v) Another alternative is to homogenize activated sludge (3-5 g
suspended solids/L). Treat the sludge in a Waring blender for 2 minutes at
medium speed. Settle the blended sludge for 30 minutes or longer if required and
decant liquid for use as inoculum at the rate of about 10 mg/L of mineral medium.
(vi) Still further reduction of the blank CO2 evolution can be achieved by
aerating the sludge overnight with CCVfree air. Use 4 mg/L activated sludge
solids as the concentration of the inoculum in the test medium when the test is
started (see paragraph (o)(13) of this guideline).
(12) Secondary sewage effluent, (i) Alternatively, the inoculum can be
derived from the secondary effluent of a treatment plant or laboratory-scale unit
receiving predominantly domestic sewage. The undiluted effluent should contain
approximately 1 x 106 microorganisms per liter. Maintain the sample under
aerobic conditions and use on the day of collection, or precondition if necessary.
The effluent should be filtered through a coarse filter to remove gross particulate
matter and the pH is measured.
(ii) To reduce its 1C content, the filtrate should be sparged with CCh-free
air for 1 h while maintaining the pH at 6.5 using orthophosphoric acid. The pH is
restored to its original value with sodium hydroxide (see paragraph (1)(6) of this
guideline) and after settling for about 1 h a suitable volume of the supernatant is
taken for inoculation. This sparging procedure reduces the 1C content of the
inoculum. For example, when the maximum recommended volume of filtered
sparged effluent (100 ml) per liter was used as inoculum, the amount of 1C
present in blank control vessels was in the range 0.4 to 1.3 mg/L (see paragraph
13
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(o)(14) of this guideline), representing 2-6.5% of test substance C at 20 mg C/L
and 4-13% at lOmgC/L.
(13) Surface waters. A sample is taken of an appropriate surface water. It
should be kept under aerobic conditions and used on the day of collection. The
sample should be concentrated, if necessary, by filtration or centrifugation. The
volume of inoculum to be used in each test vessel should meet the criteria given
in paragraph (l)(10)(ii) of this guideline.
(14) Soils. A sample is taken of an appropriate soil, collected to a depth of
up to 20 cm below the soil surface. Stones, plant remains and invertebrates should
be removed from the sample of soil before it is sieved through a 2 mm mesh (if
the sample is too wet to sieve immediately, then partially air dry to facilitate
sieving). It should be kept under aerobic conditions and used on the day of
collection. If the sample is transported in a loosely tied black polythene bag, it can
be stored at 2 to 4°C in the bag for up to one month.
(m) Test procedure—(1) Number of test vessels, (i) The number of
vessels needed for a test will depend on the frequency of analysis and the test
duration.
(ii) It is recommended that triplicate vessels be analyzed after a sufficient
number of time intervals that the 10-d window may be identified. In addition, at
least five test vessels from the test substance, reference substance and blank
control series (see paragraph (m)(3)(ii) of this guideline should analyzed at the
end of the test, to enable 95% confidence intervals to be calculated for the mean
percentage biodegradation value.
(2) Inoculated medium. Prepare immediately before use sufficient
inoculated medium by adding, for example, 2 ml suitably treated activated sludge
at 2000 mg/L to 1 liter of mineral nutrient medium (paragraph (1)(4) of this
guideline). When secondary sewage effluent is to be used, add up to 100 ml
effluent to 900 ml mineral salts medium and dilute to 1 liter with medium.
(3) Preparation of vessels, (i) Aliquots of inoculated medium are
dispensed into replicate vessels 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 (h). When using either type of inoculum, care should be taken
to ensure that the inoculated medium is adequately mixed to ensure that it is
uniformly distributed to the test vessels.
(ii) Sets of vessels are prepared as listed in paragraphs (m)(3)(ii)(A)
through (m)(3)(ii)(E) of this guideline:
14
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(A) Test vessels (denoted FT) containing the test substance;
(B) Blank controls (denoted FB) containing only the test medium plus
inoculum; any chemicals, solvents, agents or glass fiber filters used to introduce
the test substance into the test vessels should also be added;
(C) Vessels (denoted FC) for checking the procedure using the reference
substance;
(D) If needed, vessels (denoted FI) for checking a possible inhibitory
effect of the test substance containing both the test substance and reference
substance at the same concentrations as in bottles FT and Fc, respectively (see
paragraph (1)(9) of this guideline);
(E) Vessels (denoted FS) for checking possible abiotic degradation, set up
as for vessels FT but including 50 mg/L HgCl2, or sterilized by some other means
(e.g. autoclaving).
(iii) Water-soluble test substances and reference substances are added as
aqueous stock solutions to give a starting concentration of 10 to 20 mg as C/L.
(iv) Insoluble test and reference substances may be added to vessels in a
variety of ways (see paragraph (1)(7) of this guideline) according to the nature of
the substance, either before or after addition of the inoculated medium, depending
on the method of treatment of the substance. If one of the procedures given in
paragraph (1)(7) of this guideline is used, then the blank vessels FB should be
treated the same as test vessels FT but excluding the test or reference substance.
(v) Volatile test substances should be injected into sealed vessels (see
paragraph (m)(3)(vii) of this guideline) using a microsyringe. The dose is
calculated from the volume injected and the density of the substance.
(vi) Water should be added to vessels, where necessary, to give the same
liquid volume in each vessel. The headspace-to-liquid ratio (usually 1:2) and
concentration of the test substance should be such that sufficient oxygen is
available in the headspace to allow for complete biodegradation.
(vii) All vessels are then sealed, for example with butyl rubber septa and
aluminum caps. Volatile test substances should be added at this stage. If the
decrease in DOC concentration of the test solution is to be monitored and for time
zero analyses to be performed for initial 1C concentration or other determinants,
remove an appropriate sample from the test vessel. The test vessel and its contents
are then discarded.
15
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(viii) The sealed vessels are placed on a rotary shaker, with a shaking rate
sufficient to keep the contents well mixed and in suspension (e.g. 150 to 200
rpm), and incubated in the dark at 20°C ± PC.
(4) Sampling. The pattern of sampling will depend on the lag period and
rate of biodegradation of the test substance. Vessels are sacrificed for analysis on
the day of sampling, which should be at least weekly, or more frequently (e.g.
twice per week) if a complete degradation curve is required. The requisite number
of replicate vessels are taken from the shaker, representing FT, FB and Fc and, if
used, FI and Fs vessels (see paragraph (m)(3) of this guideline). The test normally
runs for 28d. If the biodegradation curve indicates that a plateau has been attained
before 28d, the test may be concluded earlier than 28d. Take samples from the
five vessels reserved for the 28th day of the test for analysis, and use the results to
calculate the confidence limits or coefficient of variation of percentage
biodegradation. Vessels representing the checks for inhibition (Fi) and for abiotic
degradation (Fs) need not be sampled as frequently as the other vessels; sampling
at day 1 and day 28 is sufficient.
(5) Inorganic carbon (1C) analysis, (i) CC>2 production in the vessels is
determined by measuring the increase in the concentration of 1C during the test.
There are two recommended methods available for measuring the amount of 1C
produced in the test, described in paragraphs (m)(6) and (m)(7) of this guideline.
Since the methods can give slightly different results, only one should be used in a
given test run.
(ii) Method-A (see paragraph (m)(6) of this guideline) is recommended if
the medium is likely to contain remnants of, for example, a glass-fiber paper
and/or insoluble test substance. This analysis can be performed using a gas
chromatograph if a carbon analyzer is not available. It is important that the vessels
should be at or close to the test temperature when the headspace gas is analyzed.
Method-B (see paragraph (m)(7) of this guideline) can be easier for laboratories
using carbon analyzers to measure 1C. It is important that the sodium hydroxide
solution (see paragraph (1)(6) of this guideline) used to convert CC>2 to carbonate
is either freshly prepared or its 1C content is known, so that this can be taken into
account when calculating the test results (see paragraph (n)(5)(i)(B) of this
guideline.
(6) Method-A: acidification to pH <3. (i) Before each batch of analyses,
the 1C analyzer is calibrated using an appropriate 1C standard (e.g. 1% w/w CC>2
in N2). Concentrated orthophosphoric acid is injected through the septum of each
vessel that is to be sampled, in order to lower the pH of the medium to <3 (e.g.
add 1 ml to 107 ml test medium). The vessels are placed back on the shaker. After
shaking for one hour at the test temperature, the vessels are removed from the
shaker, and an aliquot (e.g. 1 ml) of gas is withdrawn from the headspace of each
16
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vessel and injected into the 1C analyzer. The measured 1C concentrations are
recorded as mg C/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 CC>2
between the liquid and gaseous phases in the test vessels is 1.0 when measured as
a concentration (see paragraph (o)(13) of this guideline). This should be
demonstrated for the test system at least once as outlined in this paragraph: Set up
vessels containing 5 and 10 mg/L as 1C using a solution of anhydrous sodium
carbonate (Na2CC>3) in CO2-free water, prepared by acidifying water to pH 6.5
with concentrated orthophosphoric acid, sparging overnight with CO2-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 (m)(6)(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. If they are not, the operator
should review the procedures. This check on the distribution of 1C between liquid
and gaseous phases need not be made every time the test is performed.
(iii) If DOC removal is to be measured (water-soluble test substances
only), samples should be taken of the liquid phase from separate (non-acidified)
vessels, and the samples membrane-filtered and injected into the DOC analyzer.
These bottles can be used for other analyses as necessary, such as to measure
primary biodegradation.
(7) Method-B: conversion of COi to carbonate, (i) Before each batch of
analyses, the 1C analyzer is calibrated using an appropriate standard - for
example, a solution of sodium bicarbonate (NaHCOs) in CO2-free water (see
paragraph (m)(6)(ii) of this guideline) in the range 0 to 20 mg/L as 1C. Sodium
hydroxide solution (7M; see paragraph (1)(6) of this guideline) (e.g. 1 ml to 107
ml medium) is injected through the septum of each vessel sampled and the vessels
are shaken for 1 h at the test temperature. Use the same NaOH solution on all
vessels sacrificed on a particular day, but not necessarily on all sampling
occasions throughout a test. If absolute blank 1C values are needed at all sampling
occasions, 1C determinations of the NaOH solution will be needed each time it is
used. The vessels are removed from the shaker and allowed to settle. Suitable
volumes (e.g. 50 to 1000 jil) of the liquid phase in each vessel are withdrawn by
syringe. The samples are injected into the 1C analyzer and the concentrations of
1C are recorded. It should be ensured that the analyzer used is equipped properly
to deal with the alkaline samples produced in this method.
(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 by using 1C standards, adding alkali and
17
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equilibrating, and measuring the concentration of 1C in both the headspace and
liquid phases. The concentration in the headspace should approach zero. This
check need not be made every time the test is performed.
(iii) If DOC removal is to be measured (water-soluble test substances
only), samples should be taken of the liquid phase from separate vessels
(containing no added alkali), and the samples membrane filtered and injected into
the DOC analyzer. These vessels can be used for other analyses as necessary,
such as to measure primary biodegradation.
(n) Data and reporting - (1) Calculations, (i) Assuming 100%
mineralization of the test substance to CO2, the ThIC in excess of that produced in
the blank controls equals the TOC added to each test vessel at the start of the test,
that is:
ThIC = TOC.
The total mass (mg) of inorganic carbon (TIC) in each vessel is:
Equation [1]
TIC = (mg inorganic C in the liquid + mg inorganic C in the headspace)
= (VLxCL) + (VHxCH)
where :
VL = volume of liquid in the vessel (liter);
CL = concentration of 1C in the liquid (mg/L as inorganic carbon);
VH = volume of the headspace (liter);
CH = concentration of 1C in the headspace (mg/L as inorganic carbon).
The calculations of TIC for the two analytical methods used for measuring 1C in
this test are described in paragraphs (n)(2) and (n)(3) of this guideline. Percentage
biodegradation (%D) in each case is given by:
Equation [2]
%D = (TIG - TICh) x 100
TOC
where :
TICt = mg TIC in test vessel at time t;
TICb = mean mg TIC in blank vessels at time t;
TOC = mg TOC added initially to the test vessel.
The percentage biodegradation is calculated for the test (FT), reference (Fc) and, if
18
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included, inhibition monitoring control (Fi) vessels, from the respective amounts
of TIC produced up to each sampling time.
(ii) If there has been a significant increase in the TIC content of the sterile
controls (Fs) over the test period, then it may be concluded that abiotic
degradation of the test substance has occurred and this should be taken into
account in the calculation of %D in Equation [2].
(2) Acidification to pH <3. Since acidification to pH <3 and equilibration
(Method-A) results in the equalization of the concentration of TIC in the liquid
and gaseous phases, only the concentration of 1C in the gas phase needs to be
measured. Thus, from Equation [1] TIC = (VL + VH) x CH = VB x CH, where VB =
volume of the test vessel.
(3) Conversion of COi to carbonate. In this method (Method-B)
calculations are performed as in Equation [1], but the negligible amount of 1C in
the gaseous phase is ignored, that is VH x CH = 0, and TIC = VL x CL.
(4) Expression of results, (i) Compile a table of %D for each test (FT),
reference (Fc) and, if included, inhibition control vessel (Fj) for each day samples.
Record the amount of TIC in the blanks (vessels FB) and sterile controls (vessels
Fs).
(ii) A biodegradation curve is obtained by plotting %D against time of
incubation and if possible, the lag phase, biodegradation phase, 10-d window and
plateau phase, (phase in which the maximal degradation has been reached and the
biodegradation curve has leveled out), are indicated. If comparable results are
obtained for parallel test vessels FT (<20% difference), a mean curve is plotted
(see Fig.l); if not, curves are plotted for each vessel. The mean value of the %D
in the plateau phase is determined or the highest value is assessed (e.g. when the
curve decreases in the plateau phase), but it is important to assess that in the latter
case the value is not an outlier. Indicate this maximum level of biodegradation as
degree of biodegradation of the test substance in the test report. If the number of
test vessels was insufficient to indicate a plateau phase, the measured data of the
last day of the test are used to calculate a mean value. This last value, the mean of
five replicates, serves to indicate the precision with which %D was determined.
Also report the value obtained at the end of the 10-d window.
(iii) In the same way, plot curves for the reference substance, (vessels FC)
the sterile control (vessels Fs) and the inhibition control (vessels FI).
(iv) Calculate %D for the FI vessels, based on the theoretical 1C yield
anticipated from only the reference component of the mixture. If, at day 28,
19
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Figure 1. Biodegradation of 1-octanol in the headspace CO2 test
Q IOC
rr
I
_«
o
2C
0 ^
iKUtu ,'#v*1
*„!«< J, i I.j*
b 10
b JO
(Int«! {days}
- DFI)/DFC] x 100 > 25%, where DFC is the %D in vessels FC containing the
reference substance and DFi is the %D in Vessels FI, it may be assumed that the
test substance inhibited the activity of the inoculum, and this may account for low
values of DFx obtained under the conditions of the test. In this case the test could
be repeated using a lower test substance concentration and preferably reducing the
DIG in the inoculum and TIC formed in the blank controls, since the lower test
substance concentration will otherwise reduce the precision of the method.
Alternatively, another inoculum may be used. If in vessels Fs (abiotic check) a
significant increase (>10%) in the amount of TIC is observed, abiotic degradation
processes may have occurred.
(5) Validity of results, (i) A test is considered valid if:
(A) The mean %D in vessels Fc containing the reference substance is
>60% by the 14th day of incubation; and
(B) The mean amount of TIC present in the blank controls FB at the end of
the test is <3 mgC/L.
(ii) If these limits are not met, the test should be repeated with an
inoculum from another source and/or the procedures used should be reviewed. For
example, if high blank 1C production is a problem the procedure given in
paragraphs (1)(11) and (1)(12) of this guideline should be followed.
(iii) If the test substance does not reach 60% ThIC and was shown not to
be inhibitory (paragraph (n)(4)(iv) of this guideline), the test could be repeated
with increased concentration of inoculum (up to 30 mg/L activated sludge and
20
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100 ml effluent/L) or inocula from other sources, especially if degradation was in
the range 20 to 60%.
(6) Interpretation of results, (i) Biodegradation >60% ThIC within the
10-d window in this test demonstrates that the test substance is readily
biodegradable under aerobic conditions.
(ii) If the pass value of 60% ThIC is not attained, determine the pH value
in media in vessels that have not been made acid or alkaline; a value of less than
6.5 could indicate that nitrification occurred. In such a case, repeat the test with a
buffer solution of higher concentration.
(7) Quality assurance. To assure the integrity of data developed using
this method and to comply with current regulatory requirements, a quality
assurance program meeting EPA, FDA (United States Food and Drug
Administration), or OECD guidelines should be followed.
(8) Test Report. The test report should include the information in
paragraphs (n)(8)(i) through (n)(8)(iii) of this guideline:
(i) Test substance:
(A) Common name, chemical name, CAS number, structural formula and
relevant physical-chemical properties;
(B) Purity (and known impurities) of test substance.
(ii) Test conditions:
(A) Reference to this test guideline;
(B) Description of the test system (e.g. volume of the vessel, head space-
to-liquid ratio, method of stirring, etc);
(C) Application of test substance and reference substance to test system:
test concentrations used in the test and amount of carbon dosed into each test
vessel, any use of solvents;
(D) Details of the inoculum, including any pretreatment and
preconditioning;
(E) Incubation temperature;
(F) Validation of the principle of 1C analysis;
21
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(G) Main characteristics of the 1C analyzer employed (and any other
analytical methods used);
(H) Number of replicates.
(iii) Results:
(A) Raw data and calculated values of %D in tabular form (see paragraph
(n)(4)(i) of this guideline.
(B) The plots of %D against time for the test and reference substances,
including identification of the lag phase, degradation phase, and 10-d window
(see paragraph (n)(4)(ii) of this guideline);
(C) Percentage removal at plateau, at the end of test, and after the 10-d
window;
(D) Reasons for any rejection of the test results;
(E) Any other facts that are relevant to the procedure followed;
(F) Discussion of results.
(o) References.
(1) OECD (1992). Ready biodegradability, no. 301, adopted 17 July 1992.
Organization for Economic Cooperation and Development, Paris.
(2) Sturm, R.N. (1973). Biodegradability of nonionic surfactants:
screening test for predicting rate and ultimate biodegradation. J. A. Oil Chem.
Soc. 50, 159-167.
(3) Larson, RJ. (1979). Estimation of biodegradation potential of
xenobiotic organic chemicals. Appl. Environ. Microbiol. 38, 1153-1161.
(4) Larson, RJ, M.A. Hansmann, and E.A. Bookland (1996). Carbon
dioxide recovery in ready biodegradability tests: mass transfer and kinetic
constants. Chemosphere 33, 1195-1210.
(5) ISO 9439 (1999). Water Quality - Evaluation of ultimate aerobic
biodegradability of organic compounds in aqueous medium - carbon dioxide
evolution test. International Organization for Standardization, Geneva.
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(6) US EPA (1996). Carbon dioxide evolution test, Fate, Transport and
Transformation Test Guidelines 835.3110. U.S. Environmental Protection
Agency, Washington, DC.
(7) US EPA (1996). Aerobic aquatic biodegradation, Fate, Transport and
Transformation Test Guidelines 835. 3100. U.S. Environmental Protection
Agency, Washington, DC.
(8) Gledhill, W.E. (1975). Screening test for assessment of
biodegradability: linear alkyl benzene sulfonate. Appl. Microbiol. 30, 922-929.
(9) Weytjens, D., I. Van Ginneken and H.A. Painter (1994). The recovery
of carbon dioxide in the Sturm test for ready biodegradability. Chemosphere 28,
801-812.
(lO)Ennis, D.M. and A. Kramer (1975). A rapid microtechnique for
testing biodegradability of nylons and polyamides. J. Food Sci. 40, 181-185.
(ll)Ennis, D.M., A. Kramer, C.W. Jameson, P.H. Mazzocchi and WJ.
Bailey (1978). Structural factors influencing the biodegradation of imides. Appl.
Environ. Microbiol. 35, 51-53.
(12) Boatman, R.J., S.L. Cunningham and D.A. Ziegler (1986). A method
for measuring the biodegradation of organic chemicals. Environ. Toxicol. Chem.
5,233-243.
(13) Struijs, J. and J. Stoltenkamp (1990). Head space determination of
evolved carbon dioxide in a biodegradability screening test. Ecotox. Environ.
Safe. 19,204-211.
(14) Birch, R.R. and RJ. Fletcher (1991). The application of dissolved
inorganic carbon measurements to the study of aerobic biodegradability.
Chemosphere 23, 507-524.
(15) ISO 14593 (1999). Water Quality - Evaluation of ultimate aerobic
biodegradability of organic compounds in an aerobic medium-method by analysis
of inorganic carbon in sealed vessels (CO2 headspace test) International
Organization for Standardization, Geneva.
(16) Battersby, N.S. (1997). The ISO headspace CO2 biodegradation test.
Chemosphere, 34, 1813-1822.
(17) Battersby N.S., D. Ciccognani, M.R. Evans, D. King, H.A. Painter,
D.R. Peterson, and M. Starkey (1999). An inherent biodegradability test for oil
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products: description and results of an international ring test. Chemosphere 38,
3219-3235.
(18)OECD (1988). OECD Ring-test of methods for determining ready
biodegradability: Chairman's report (M. Hashimoto; MITI) and final report (M.
Kitano and M. Takatsuki; CITI). Organization for Economic Cooperation and
Development, Paris.
(19)ASTM (1993). Annual Book of ASTM Standards, Volumes 11.01
and 11.02 on Water and Environmental Technology, and Volume 14.02 on
General Methods and Instrumentation. American Society for Testing and
Materials, Philadelphia, PA.
(20) OECD (1984). Activated sludge, respiration inhibition test, no. 209,
adopted 4 April 1984. Organization for Economic Cooperation and Development,
Paris.
(21) Struijs I, MJ. Stoltenkamp-Wouterse and A.L.M. Dekkers (1995). A
rationale for the appropriate amount of inoculum in ready biodegradability tests.
Biodegradation 6, 319-327.
(22)WRC (1999). Ring-test of the ISO headspace CO2 method:
application to surfactants: surfactant ring test-1, Report EU4697. Water Research
Centre, May 1999, Medmenham, UK.
(23) Richterich, K. and J. Steber (2001). The time-window - an
inadequate criterion for the ready biodegradability assessment of technical
surfactants. Chemosphere 44, 1649-1654.
(24) ISO 10634 (1996). Water Quality - Guidance for the preparation and
treatment of poorly water-soluble organic compounds for the subsequent
evaluation of their biodegradability in an aqueous medium. International
Organization for Standardization, Geneva.
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