United States      Prevention, Pesticides      EPA712-C-08-001
         Environmental Protection   And Toxic Substances       October 2008
         Agency        (7101)
4>EPA   Fate, Transport and
         Transformation Test

         OPPTS 835.3140
         Ready Biodegradability
         —CO2 in Sealed Vessels
         (Headspace Test)

      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.

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

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

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

       1C:  Inorganic carbon

       Inherent Biodegradability: A classification of chemicals for which there is
unequivocal evidence of biodegradation (primary  or  ultimate) in any  test  of

       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

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%

       (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

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

       (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.

       (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)
Mean Percentage
Coefficient of variation
Number of
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.

Table 2. Summary of results from
(see paragraph (o)(22) of this c
Test Substance
Mean Percentage
EU ring test
Coefficient of
Number of
   * 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
   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

       (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.

       (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

(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

   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:

       (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

       (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

       (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

and  reference  substance,  each  at  the  same  concentration  as when  present

       (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

       (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

       (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.

       (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

(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

       (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:

       (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

       (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.

       (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

       (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

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

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

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

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

       (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,

       Figure 1. Biodegradation of 1-octanol in the headspace CO2 test
         Q   IOC
              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

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

       (E) Incubation temperature;

       (F) Validation of the principle of 1C analysis;

       (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

       (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.

       (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,

       (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.

       (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

products: description and results  of an international ring test. Chemosphere 38,

       (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,

       (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.