Biodegradabilidad Oxitop y Aplicaciones

Biological Degradability:
Determination by
simplified manometric
measuring methods
1st Symposium
26th September 2000
Ludwigshafen
Wissenschaftlich-Technische
Werksttten GmbH & Co. KG
Dr.-Karl-Slevogt-Strae 1
D-82362 Weilheim
Tel 0881 / 183-0
Fax 0881 / 62539
E-Mail Info@WTW.com
Internet www.WTW.com
The significance of standardized degradation tests...
The significance of standardized

degradation tests in the testing of
substances
Dr. Udo Pagga, BASF AG Ludwigshafen
1. Biological degradation in the environment

Substance and energy cycles play an important role in ecology, in
particular the cycle of carbon that begins with the build up of organic
matter of carbon dioxide and water by producers (plants), goes
through the utilization of the nutrient formed by this and is continued
by consumers (animals). The entire resulting organic matter, including
the biomass of the plants and the animals with their excreted products
is eventually decomposed again into low molecular basic substances
or into stable degradation products (catabolites) (Fig.1) by the
destruents, particularly bacteria and fungi. This biological degradation
takes place in the environment in multifarious forms because the
destruents have had millions of years in which to find efficient
methods of biochemical degradation for the realization of their food.
However, in addition to natural substances, many anthropogenic
substances produced by man that have only been introduced into the
environment in large amounts since the beginning of industrialization,
are also biologically degradable because their structure often
resembles that of natural substances.
Sun
Fig. 1:
Substance and energy cycles
= light energy Consumers
= animals
Organic
substances
Producers
= plants Anorganic
substances
Destruents
= microorganisms
1st Symposium on Biological degradability.. 26th September, 2000 Theme 1: Dr. Udo Pagga
The most important biological degradation processes in the natural

environment need oxygen (aerobic degradation), but anoxic and
anaerobic degradation processes that take place with the exclusion of
oxygen also play an important role in environmental compartments
(Fig. 2). For efficient degradation to take place, some important
conditions must be met. These include, for example, an adequate
supply of nutrients (e.g. nitrogen and phosphorous compounds),
vitamins and trace elements (some heavy metals) or an adequate
buffering of the environment as the basis of a favorable pH range.
Very important for biological processes is the basic presence of water.
Since the transport and distribution of substances is also quite possible
in an aqueous environment, aerobic degradation in aquatic
environmental systems is of exceptional significance. In streams,
rivers, ponds or lakes, the best conditions are always found where the
suitable colonization sites are available for the microorganisms, e.g.
on stones or water plants on the bank area where plant life can form
and, as a result, close contact attained with the substances to be
degraded.
Biological degradation processes take place not only in fresh water,

but also in marine environments. Apart from the much higher salt
content that enables only the specialized, so-called haloform
microorganisms to survive and, consequently, degradation processes
to take place, there are extremely low concentrations of the substances
and microorganisms that in particular constitute the special features of
marine degradation. By contrast, extremely high concentrations of
degradable substances as well as a wide variety of microorganisms
can be found in many terrestrial environment compartments such as
humus-rich arable land or sediments.
Fig. 2 :
Aerobic and anaerobic degradation
processes
Aerobic degradation organic substance + O2 -----------------> CO2 + H2O + biomass
Anaerobic degradation organic substance ------------------------> CO2 + CH4 (biogas) + biomass
The forms of colonization and activities of man have, over a long

period of time, led to specific local pollution of the environment.
Since the beginning of the 20th century, natural degradation processes
have been implemented in technical plants and used for the disposal of
wastewater and refuse. Biological wastewater treatment plants with
aerobic sedimentation basins and sludge towers for anaerobic sludge
stabilization are still the most efficient facilities for cleaning
wastewater. Aerobic composting works or anaerobic fermentation
plants are being used increasingly for the treatment of vegetation and
bio-refuse. The same biological degradation processes that take place
in the natural environment take place instead in these technical plants,
although under optimized and controlled conditions.
Industrialization has also increasingly resulted in the release of

anthropogenic substances into the environment, e.g. the products of
the chemical industry. From around the second half of the 20th
century, this has led not only to problems in the treatment of
wastewater such as, e.g. the large amounts of foam in rivers and
wastewater treatment plants as a result of poorly degradable or so-
called hard detergents, but also to the necessity of finding out more
about the behavior of substances in the environment. Laboratory
methods were developed to predict the whereabouts, the degradation,
the distribution and the eco-toxic effect of the different substances.
Initially, this was only done for plant agents and detergents, but since
around 1980 this has also included the vast number of other
chemicals. Because a substance that is being degraded, and
consequently is no longer present in the environment, presents no
long-term ecological threat, the test for biological degradability
together with the analysis of eco-toxic effects and bio-accumulation is
the most important criterion in the ecological evaluation of substances
and products.
2. Test of biological degradability

Degradation tests in the laboratory can only lead to a practicable
prediction of the degradation behavior in the environment if essential
aspects of the respective environmental compartments are taken into
account in the test. The same parameters that effect biological
degradation in the environment or in technical plants also have an
effect in laboratory test systems. An indispensable prerequisite of
biological degradation are suitable microorganisms that are specified
as inoculum in the test preparation, usually in the form of mixed
inoculum that was taken from the environment. The inoculum cannot
be standardized and also the use of pure cultures is not practical for
standard degradation analyses. Only the origin and any possible
preliminary treatment or preadaptation of the substance to be tested
can be defined. Mostly, activated sludge or the effluent of a
wastewater treatment plant is used although, depending on the test,
surface water, seawater, sludge, sediment or soils can also be used.
The influencing variables include the molecular structure of the test
substance, its bio-availability, the concentration in the test or,
depending on the type of test, the absence or presence of other
degradable substances to enable a so-called cometabolic degradation
in the latter case. Furthermore, aerobic tests require adequate
ventilation and mixing whereas anaerobic tests require the strict
exclusion of oxygen. Other important factors are the composition of
the nutrient medium, the pH value, the test temperature, the duration
of the test, the type of sampling, the sampling frequency and, of
particular significance, the measured analysis parameters.
The degradation results are decisively shaped by the choice of the
analytical measurement parameters. To determine the complete
degradability (mineralization, ultimate biodegradation), so-called sum
parameters are usually used. The most important of these are the
decrease in the dissolved organic carbon (DOC) in comparison to the
initial concentration, the biogenic production of carbon dioxide (CO2)
in comparison to the theoretical CO2 production (ThCO2) and the
biochemical oxygen demand (BOD) in comparison to the calculated
theoretical reference value (ThOD). For metrological reasons, in the
use of sum parameters, the test concentrations of individual substances
are much higher than the real environment concentrations in
wastewater or even in water itself and, thus, it also takes a relatively
long time until the degradation process in the test is completed. For
the determination of primary degradation that is defined as the loss of
the material identity of the test substance, substance-specific analysis
methods such as gas chromatography (GC) or high pressure liquid
chromatography (HPLC) are required. If degradation tests are required
within the trace range, the use of radioactively marked compounds
(mostly C14) is the method of choice.
For a simple batch test, the samples are prepared in suitable test
vessels. The test samples are made up of the test substance, an
inorganic nutrient medium and the inoculum. The test begins by
switching on the stirrer and the ventilation. The intended analysis
parameter is either measured directly, or in part even measured
continuously or in samples that are taken at regular intervals. The
measured values are plotted against the duration of the test to create a
degradation curve. Typical degradation curves exhibit a lag phase in
which the inoculum adapts itself to the test substance, a degradation
phase in which the actual substance conversion results and a plateau
phase when the degradation process is complete. At the end of the test,
usually when a distinct plateau phase has been reached, the degree of
degradation as compared to a defined initial value or reference value
is determined as a percentage. Depending on the test method and the
problem, other data such as the slope of the degradation curve can also
be determined as a variable of the degradation kinetics (Fig. 3).
The duration of the test using standard methods is relatively short
compared with the periods of time in which substances occur in the
environment. However, on the other hand, the test period is also
considerably long if the usual average residence time of wastewater in
wastewater treatment plants that is in the range of hours is taken for
comparison. Most aerobic batch tests take 28 days whereas continuous
tests take 12 weeks or longer. Anaerobic, marine or terrestrial tests
can take even longer, e.g. the degradation tests for polymers which
can take up to half a year. The standard test duration of approximately
one month is practical because the entire lag phase that may be several
days in a non-adapted inoculum lies within this time period. Due to
the relatively high test concentration of the test substance, the
degradation phase must also be long enough as test concentrations that
can be 100 to 1000 times greater than in the environment also require
more time for complete degradation, even if the degradation rates
correspond to those of the environment. Finally, an appropriate length
of the plateau phase must also lie within the test period in order for the
degree of degradation to be determined with any degree of reliability.
Fig. 3:
Degradation curve
DOC degradation (%)
100
90
80 Degradation phase
Lag phase Plateau
70 h
60
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Test period (d)
In addition to the degradation of organic substances, there are also

other bacterial activities that are of great significance in the
environment, such as nitrification, denitrification, nitrogen fixing,
conversion processes in the sulfur, iron or manganese cycle as well as
autotrophic carbon dioxide fixing. Such special problems each require
special methods. However, with the exception of various test methods
for the determination of the inhibitory effect on bacteria, mostly no
standardized methods exist.
3. Standardized degradation test methods

Degradation tests are carried out using standardized test methods that
exist either in the form of OECD Guidelines for Testing of Chemicals,
as almost identical guidelines in the EU guidelines for hazardous
substances or as international (ISO), European (EN) or national (DIN)
norms. The conventional test methods can be organized according to
the following criteria:
1) Aquatic (waters, wastewater treatment plants) and terrestrial
(soils, compost);
2) Static (batch test), semi-continuous and continuous;

3) Tidal and marine;
4) Aerobic and anaerobic;
5) Sum parameters (DOC, BOD, CO2, biogas) for the determination
of the mineralization or use of substance-specific analysis
methods for primary degradation;
6) Use of radioactively marked compounds (mostly C14) for the
degradation analysis of low concentrations or for special problems
(e.g. identification of metabolites);
7) Tests for biological degradability and elimination tests of water;
8) Tests for the determination of the easy (ready) and potential
(inherent) degradability and simulation tests.
In a typical aerobic, aquatic batch test of easy biological degradability,

the test substance is specified as the single source of carbon in a
concentration of between 20 and 100 mg/l in a defined dilution water.
After neutralization, the activated sludge of a municipal wastewater
treatment plant is injected in a concentration of 30 mg/l dry substance.
The following test preparations are required as a rule to obtain a valid
test result and to differentiate between biological degradation and
abiotic elimination mechanisms such as adsorption or stripping:
1) Preparations with test substance, inorganic medium and inoculum;
2) Blank value preparations with only inoculum and inorganic
medium;
3) An activity control with inoculum, inorganic medium and a
known reference substance (e.g. aniline);
4) An inhibition control with inoculum, inorganic medium, test and
reference substance. This is required in order to determine
whether the test substance inhibits the known degradation of the
reference substance and consequently to preclude that insufficient
degradation is the result of a toxic effect.
5) A preparation with test substance and inorganic medium, but
without inoculum to detect abiotic, physical-chemical elimination
mechanisms;
The preparations are shaken or stirred for the specified period of the
test at constant room temperature (20-25o C) and ventilated at the
same time. Samples are taken and analyzed at regular intervals or,
depending on the test, at the end of the intended incubation period. In
some tests such as the respirometric degradation test, the degradation
parameters are measured continuously. In this case of oxygen
consumption, measurement is made either via a decrease in pressure
or an additional supply of oxygen. As the test method for wastewater,

the biochemical oxygen demand for 5 days (BOD5) is also used that is
then compared with the chemical oxygen demand COD to determine a
percentage amount of degradation.
In the anaerobic degradation test, the sample is incubated in a closed,
gasproof test vessel at 35o C for up to 45 days. Degradation parameters
include the biogas determined by a pressure measurement and the
amount of inorganic carbon (dissolved CO2 and carbonate) present at
the end of the test.
In the semi-continuous test method (SCAS test), the nutrient medium
contains a synthetic or real wastewater as the organic substrate. The
test preparations are prepared in the same way as a batch test, but,
after an incubation period of one day, the ventilation is switched off
and the supernatant liquid of deposited inoculum is decanted. Fresh
medium and new test substance are added and the ventilation is started
again. The DOC of the decanted liquid is determined and is used for
the determination of the amount of degradation.
Test methods that simulate the degradation in wastewater treatment
plants or rivers run continuously. In the activated sludge simulation
test that, in principle, represents a mini wastewater treatment plant
with sedimentation and a final settling tank, the test substance and the
organic medium have to be dosed continuously and the decrease of the
DOC in the inlet and outlet of the plant is measured. However, this is
only possible if at least two plants are operated in parallel, one
containing only the organic medium and one containing the medium
and the replenished test substance. For example, in the continuous
flow test, a natural pond water is directed through a cascade with
several basins and the test substance is added in low, realistic
concentrations. The test concentration is measured in the individual
basins over several days using substance-specific detection methods or
via C14-marked compounds.
An overview of the most important standardized test methods that are
given in Appendix 1 is given in the ISO norm 15462 and the
publication U. Pagga Testing Biodegradability with Standardized
Methods in Chemosphere 35, 2953-2972 (1997).
4. Evaluation of the results and consequences of degradation

testing
An overview of the evaluation of results from degradation tests are
given in Table 1.
Table 1: Evaluation of the test results
DOC tests BOD/CO2 tests Evaluation
>70% in 10 days >60% in 10 days readily biologically degradable (acc. to OECD criteria)
<70% in 10 days <60% in 10 days not readily biologically degradable (acc. to OECD criteria)
>70 % >60 % biologically degradable and, acc. to OECD criteria,

potentially fully degradable
20-70 % 20-60 % moderately or partly biologically degradable under the test

conditions
>20 % >20 % indications of potential degradability acc. to OECD criteria
<20 % <60 % poorly biologically degradable under the test conditions
If, in the use of the DOC as an analysis parameter, no unequivocal

indications are present that this involves biological degradation, e.g. if
the degradation curve shows no clear lag phase, a statement can only
be made on the elimination from the water. The biochemical oxygen
demand in comparison to the theoretical reference value is a clear
measurement parameter for the biological degradation just like the
production of carbon dioxide.
In the determination of readily biological degradability, a limit of 70%
DOC decrease must be reached within 10 days. As the
microorganisms only oxidize a part of the organic test substance and
the rest is converted into biomass, the tests that use CO2 and BOD as
measurement parameters already exhibit sufficient degradation at
60%. Both the limit of 70% as well as the limit of 60% appear to be
relatively low for the evaluation of a complete degradation. This is
however quite sensible as the OECD criteria for pure substances apply
and it can be assumed that the degradation also continues beyond the
period of the so-called 10-day window, or at least will be complete in
the environment. In the real environment, the substance degradation is
always better and more complete as a rule than under the restrictive
conditions of the tests for readily degradability. At the same time, it is
accepted that, in rare isolated cases, a wrongly positive statement can
also result that classifies a substance as readily degradable although a

stable catabolite was formed and also the degradation in the
environment is not complete.
The OECD criteria for readily degradability also include the use of an
inoculum that was not preadapted as well as the limits and the 10-day
window. If a test substance is classified as readily biologically
degradable (readily biodegradable), experience indicates that usually
rapid and complete degradation also takes place in the environment, in
particular in wastewater treatment plants and surface waters.
If insufficient degradation was measured in a test for readily
degradability, the test substance can be nevertheless potentially
degradable (inherently biodegradable). This must be proved by further
investigations in a suitable test. These tests indicate potential
degradability, e.g. higher inoculum concentrations, a longer duration
of the test and better adaptation possibilities for the inoculum. If a test
substance is degraded under these conditions, it can be assumed that it
is not persistent and sooner or later will also degrade in the
environment. In adapted wastewater treatment plants, this can take
place very rapidly, i.e. within the average residence time of the
wastewater. If a test substance is not sufficiently degradable or cannot
be eliminated under these test conditions, it can be assumed that this
would also be the case in the environment and in wastewater treatment
plants. A substance cannot be both absolutely easily degradable and
still potentially degradable at the same time.
If more, or more precise, information is required on the degradation
behavior of substances in the environment, a simulation test may be
required such as, for example, an activated sludge simulation test for
more precise predictions of the degradation in biological wastewater
treatment plants or a batch test with flowing water or the continuous
flow test for the degradation in surface water.
The ecological quality of environmental compartments, substances
and products is regulated in numerous international, European and
national guidelines, laws, decrees and administrative directions. This
also includes the results from degradation tests in a huge number of
evaluations and often has far-reaching consequences. Examples are
the registration of new substances according to chemical law,
guidelines of the EU regulation on scrap material and the guidelines
and laws on the protection of soil and water or the identification of the
environmental danger of substances and preparations according to the
hazardous material regulation or the award of an ecological label such
as the blue angel. The degradation is a factor for the classification in
water hazard classes with the resulting technical consequences for the
storage and transport of substances and products. Sufficient
degradation is the prerequisite for the licensing of washing powders
and cleaning agents as well as the classification of compostable
plastics and packagings. The chemical industry publishes the data
provided in safety datasheets, in scrap material reports or enters them
in a European database (IUCLID).
A further example for the consequences that result from the

degradation testing of a substance is the assessment of the
environmental risk. To perform a "risk assessment", the ecological-
toxicological information on a substance are collected from analyses
and evaluated and a "predicted no effect concentration" (PNEC)
determined from them on the one hand. On the other hand, the
environmental behavior of the substance is determined from
measurements or test data and a probable environmental concentration
the "predicted environmental concentration" (PEC) is derived. PEC
and PNEC are compared with one another and the conclusion is
reached that an environmental risk is present if the PEC is greater than
the PNEC. Lower environmental concentrations, i.e. a lower PEC, can
be expected in a readily degradable substance than in a persistent or
badly degradable substance.
Degradation results are also used to make predictions on the
degradation and elimination behavior of wastewater in wastewater
treatment plants. In this case, the complex and often varying
composition of the test material wastewater must be taken into
account. Chemical reactions, complex formation, stripping processes,
precipitations and adsorption of individual components or different
solubilities of varying pH values can occur and lead to abiotic
elimination processes that hamper an unequivocal interpretation of the
results. For wastewater, the BOD/COD ratio is still often used for the
evaluation of degradability. Particularly because the conditions of the
dilution BOD are very restrictive and the oxygen measurement
records only form part of the wastewater components that is
biochemically oxidized, not however the part that is converted into
new biomass, a BOD5/COD ratio of >50% already reveals a largely
complete biological degradation. The limit for an adequate
degradation lies at a >80% decrease in the DOC for the static test and
for the decrease in DOC test as well as in the activated sludge
simulation test. Such a degree of cleaning is also usually achieved in
well-run real wastewater treatment plants.
5. Quality management and supply source of method regulations

To improve the quality of the analyses, to guarantee their
understandability even after several years and to reach unrestricted
worldwide acceptance of the results, substance tests are usually
performed according to recognized quality criteria. These are, e.g. the
OECD principles of Good Laboratory Practice (GLP) that are
contained in the German chemical law. Experience has shown that the
GLP in particular finds high acceptance by authorities and is not only
indispensable for substance registration according to the chemical law.
However, the accreditation of a laboratory according to the principles
of the norm ISO/IEC 17025 Allgemeine Anforderungen an the
Kompetenz von Prf- and Kalibrierlaboratorien at one of the
departments recognized by the Deutschen Akkreditierungsrat (DAR)
is also a good basis for the laboratory.
10
DIN, EN and ISO norms can be obtained from Beuth Verlag GmbH,
Burgrafenstr. 6, 10772 Berlin. ISO norms are also available from the
International Organization for Standardization, Case Postale 56 CH-
1211 Genf 20 (Switzerland). Degradation methods are described in the
Anhang zur EG-Richtlinie 88/302/EWG vom 18. November 1987 -
Amtsblatt der Europischen Gemeinschaften L 133, 31.Jahrgang,
30.Mai 1988 and in the Anhang zur EG-Richtlinie 92/69/EWG vom
31.Juli 1992 - Amtsblatt der Europischen Gemeinschaften L 383 A,
35.Jahrgang, 29. Dezember 1992, Vertrieb ber Bundesanzeiger,
Postfach 108006, 5000 Kln 1. OECD Guidelines for the Testing of
Chemicals can be obtained from the Organisation for Economic
Cooperation and Development, 2, rue Andre Pascal, F-75775 Paris
Cedex 16 1993.
11
Appendix 1
Overview of standardized methods for biological degradation
Degradation test OECD EG ISO DIN EN ISO

DOC decrease test 301 A 92/69/EWG C.4-A 7827 7827
Modified OECD screening test 301 E 92/69/EWG C.4-B - -
CO2 development test 301 B 92/69/EWG C.4-C 9439 9439
CO2/DOC combined test 9439 App. D
Manometric respiration test 301 F 92/69/EWG C.4-D 9408 9408
MITI test 301 C 92/69/EWG C.4-F - -
Two-phase closed bottle test 10708
Closed bottle test 301 D 92/69/EWG C.4-E 10707 10707
Zahn-Wellens test 302 B 88/302/EWG 9888 9888
SCAS test 302 A 88/302/EWG 9887 9887
Simulation test with activated sludge 303 A 88/302/EWG 11733 11733
Anaerobic degradation test 11734 11734
Preparation of substances that are hard 10634 10634
to dissolve in water
Degradation at low concentrations 14592
Parts 1 and 2
CO2 head space test 14593
Test overview of degradation tests 15462
Marine degradation tests 16221
Adsorption on activated sludge 18749
Respirometer (plastic) 14851 DIN 54900/2
CO2 test (plastic) 14852 DIN 54900/2
Anaerobic degradation in the water 14853
(plastic)
Aerobic composting test (plastic) 14855 DIN 54900/2
Anaerobic high solid test (plastic) 15985
Test schedule of comp. plastic 15986 DIN 54900
Disintegration test (plastic) 16929 DIN 54900
Degradation in soil (plastic) 17556
12

Manometric respiration tests in the OxiTop Control test system
Carrying out the manometric

respiration test in the OxiTop Control
test system
Dr. Peter Reuschenbach, BASF AG Ludwigshafen
1. The search for a new type of respirometer

The laboratory for experimental toxicology and ecology of the BASF
tests approximately 260 pure substances and substance mixtures each
year for their biological degradability using standardized test methods.
In approx. 25 % of cases the test is performed using the
manometrically controlled respirometer, usually with the application
of the Good Laboratory Practice (GLP). Above all, sparingly water-
soluble substances and volatile substances are tested in the
manometric respiration test.
The measuring system that has been routinely employed in the BASF
for more than 25 years is the Sapromat of the Voith company of
Heidenheim or the IBUK company of Knigsbrunn (Fig. 1). Due to its
long working life in the laboratory, very extensive experience has
been gained with this type of instrument. In addition, the test
apparatus works extremely precisely and provides reliable and
reproducible results. As a result, the degradation data determined in
the Sapromats are accepted without criticism by both the clients of the
substance analyses as well as by the authorities.
Fig. 1: Sapromat,
test apparatus for the manometric respiration
test
1st Symposium on Biological degradability.. 26th September, 2000 Theme 2: Dr. Peter Reuschenbach

Because, however, the Sapromats are relatively old in their basic

procedural concept, a new type of respirometer, the OxiTop Control
test system of the company WTW in Weilheim (Fig. 2), was to be
tested as to its practicability for degradation studies.
Numerous comparison tests were now performed to investigate
whether the new test system fulfilled the OECD validity criteria for
tests of easy biological degradability and to what extent the
degradation graphs for various substances determined in the two types
of respirometer agree.
2. The manometric respiration test

Both the Sapromat and the OxiTop Control test system use activated
sludge from a laboratory wastewater treatment plant that is operated
using municipal wastewater, an organic carbon compound and the
necessary nutrient salts incubated in closed vessels at room
temperature for a month. In addition to test substance preparations,
blank value and reference preparations were also tested.
Fig. 2:
OxiTop Control test system, the test
apparatus for the manometric respiration test
The measurement of biological degradability in two types of

respirometer is based on the fact that in the oxidation of organic
carbon compounds by the bacteria from the activated sludge oxygen is
consumed and carbon dioxide is formed, which is absorbed by a
suitable absorber. This creates negative pressure in the closed systems.
This negative pressure is used by the OxiTop Control test system for
calculating the biochemical oxygen demand (BOD). The test

apparatus has no system for the subsequent supply of oxygen. Hence,

the amount of carbon called for must be dosed so that a problem-free
supply of oxygen is possible from the gas atmosphere above the
culture liquid of a preparation.
In contrast, any negative pressure in the Sapromats, registered by a
simple pressure indicator, causes the instrument to switch on an
electrolysis cell that produces oxygen from a copper sulfate solution.
The oxygen production runs until the original pressure is restored in
the system. The amount of current consumed by this process is
registered. It is a measure of the biochemical oxygen demand.
To obtain information on the degree of degradation of a substance, the
BOD value is compared with a reference value. On the one hand, the
theoretical oxygen demand (ThOD) can be used for this, or on the
other hand, the measured chemical oxygen demand (COD) can be
used. If, in the course of a test that takes 28 days, the biochemical
oxygen demand of a substance rises from 10 % of the ThOD to at least
60 % of the ThOD within ten days, the substance can be classified as
readily biologically degradable according to the OECD evaluation
criteria.
Table 1 contains a compilation of the most important test conditions
when performing the manometric respiration test in the two types of
respirometer.
Tab. 1: Compilation of the test conditions when performing the
manometric respiration test in the Sapromat and OxiTop
Control test systems
Sapromat OxiTop Control
test system
Test vessel (ml) 500 510 (1)
Culture volumes (ml) 250 164 (1)
Supply of depleted oxygen yes no
Amount of oxygen/preparation (mg) unlimited 83
Maximum BOD (mg/l) unlimited 508
Carbon dioxide absorber soda lime NaOH pellets
Pretreatment of the inoculum:
- Washing steps in demineralized H2O (n) 1-2 1-2
- Consumption time (days) 1-2 1-2
Inoculum (mg TS/l) 30 30
Nutrient salts OECD medium OECD medium
Substance concentration (mg/l) 100 100
o
Temperature ( C) 22 1 22 1
Duration of the trial (days) 28 28
(1)
Setting of the BOD measurement range in the OxiTop Controller OC 110 (mg/l): 0-
400

3. The biochemical oxygen demand in blank value preparations

and test substance preparations in the comparison of the
Sapromat / OxiTop Control test systems
3.1 The compliance with the OECD validity criteria with respect
to the biochemical oxygen demand in blank value
preparations
According to the validity criteria of the OECD test regulations of the
manometric respiration test, after 28 days the BOD value of blank
value preparations usually lies within a range of between 20 - 30 mg
O2/l and should not exceed 60 mg O2/l.
The analysis of 161 blank value preparations performed in the
Sapromat over eight years resulted in the following picture: At the end
of the trial after 28 days the BOD value lay in the middle at 20 6.4
mg O2/l (Fig. 3). In contrast, noticeably higher values were measured
in the OxiTop Control test system with average values of 35.0 11.1
mg O2/l in 22 degradation tests. In fact, the average value lay above
the limit range of 20 - 30 mg O2/l although, however, the blank value
preparations fulfilled the validity criteria of the OECD standard test.
As can be seen from Figure 3, the BOD in the blank value
preparations increased in the OxiTop Control test system more
quickly from the beginning of the test than in the Sapromat.
Fig. 3:
Development of the biochemical
oxygen demand (BOD) in blank value
preparations during the manometric
respiration test

3.2 The graph of the biochemical oxygen demand in blank value

and test substance preparations using the example of a
degradation test with aniline
Using aniline as the test substance demonstrated the typical
differences that occur in degradation tests performed at the same time
in the two different types of respirometer. To the extent that the BOD
values of the blank value preparations (Fig. 4a) of the test systems
develop at different rates during the course of a test, the BOD graphs
of the test substance preparations also behave differently (Fig. 4b).
After 28 days, the difference that occurred in the blank value
preparations of the respirometer approximately corresponds to the
value measured in the test substance preparations. However, the
differences between the two types of respirometer cancel each other
out again in the calculation of the biological degradation by the
subtraction of the respective blank values from the BOD values of the
test substance preparations. Consequently, the degrees of degradation
determined in the two types of test apparatus almost agree in the
plateau phase (Fig. 4c).
4a 4b 4c
Fig. 4 a-c: Degradation of aniline in the manometric respiration test in the comparison of the Sapromat /
OxiTop Control test systems
4a: Graph of the biochemical oxygen demand in the blank value preparations
4b: Graph of the biochemical oxygen demand in the test substance preparations
4c: Biological degradation of aniline

3.3 The compliance with the OECD validity criteria with respect
to the deviation of parallel test preparations from one
another
According to the OECD criteria, the rate of degradation in parallel
preparations of a test may not deviate from one by more than 20 %
from one another at the end of the test, in the plateau phase or at the
end of the 10-day window. The compliance with this validity criterion
was checked in the OxiTop Control test system using substances
whose biological degradability is evaluated varyingly according to the
OECD criteria. Benzoic acid was tested as a typical example of a
biologically easily degradable chemical and, BASF 980803 was tested
as an example of biologically degradable substances (60 % in 28
days). BASF 990020 proved to be moderately or partly degradable (20
60 % in 28 days; Fig. 5) in the degradation test.
The percentage deviation within the parallel preparations of a test lay
below 20 % for the substances tested (Fig. 5). At the same time, it
confirmed an observation also made in other test procedures that,
usually the deviation in biologically easily degradable substances is
particularly low, while it is frequently higher in less easily degradable
compounds.
Fig. 5:
Analyzing the deviation of parallel
preparations in degradation tests using the
OxiTop Control test system

4. The search for a reference substance for the

OxiTop Control test system
4.1 The reference substance aniline

The laboratory for ecological tests of the BASF uses aniline almost
exclusively as the reference substance in degradation tests. Aniline
was selected because its degradation shows a sigmoid curve typical of
microbiological degradation processes with a clearly pronounced lag,
exponential, and stationary phase and stretches over approximately
half of the trial time. The substance is water-soluble and, as a result,
analytically easily available. It absorbs practically none of the
activated sludge and the stripping in the standard test with active
depth gas dispersion can be ignored. Technical, organizatorial and
personal safety measures ensure that the laboratory employees do not
come directly into contact with the substance that is classified as toxic
and environmentally hazardous. Workplace concentration
measurements also verify that by complying with the safety measures
the allowable limit for aniline at the workplace is clearly undercut and,
thus, the handling of the substance presents no danger to the
employees.
4.2 Differences in the degradation of aniline in the most

important standard test for checking the easy biological
degradability of substances
The degradation of aniline in the most important standard test for
ready biological degradability of substances does not run uniformly,
but can be divided into two groups where the respective analysis plays
an important role (Fig. 6): Together with the CO2 development test
(modified Sturm test) and the CO2 headspace test, the manometric
respiration test forms a group of tests whose analysis is based on a
biological principle and only records degradable substances such as
aniline. While the manometric respiration test measures the amount of
oxygen that is consumed in the biological oxidation of aniline, the two
other tests register the amount of CO2 that is created by the
degradation of the substance. Within this group, the degradation in the
CO2 headspace test takes place somewhat more quickly (primarily in
the first seven days) than in the two other degradation tests (Fig. 6).
However, a detailed statement is not possible because of the low
sample count in the CO2 headspace test.
The degradation in the Die Away test is observed by means of carbon
measurements in the supernatant liquid (DOC analysis; dissolved
organic carbon). The elimination of aniline in this test clearly differs
from the CO2 development test and the CO2 headspace test in the
manometric test. Not only is the shorter duration of the lag phase
conspicuous, but also the steeper increase of the degradation in the
exponential phase. The degree of degradation in the plateau phase is
approximately 95 % and, as a result, surpasses the other methods by
approx. 10 - 15 %. The plateau phase started on average

approximately a week earlier in this test than, e.g. in the manometric

respiration test (Fig. 6).
Although not absolutely specified, parallel to the CO2 development,
the DOC elimination during the degradation of aniline was also
observed in some tests of the CO2 headspace test.
The DOC elimination graph of this test runs approximately parallel to
the graph of the Die Away test. Also the static test (Zahn Wellens
test), that in fact does not belong to the group of tests for easy
biological degradability as a result of the higher activated sludge and
test substance concentration, confirmed the very rapid and early DOC
elimination of aniline from the aqueous phase (Fig. 6).
Thus, the degradation of aniline in standard test methods can be
described by the following summary: First, there is a very rapid
assimilation of the substance by the activated sludge as shown by the
DOC elimination tests. Subsequent to this, the biological oxidation of
the substance starts after a time delay as can be seen from the oxygen
depletion in the Sapromat or from the CO2 formed in the CO2
development test and CO2 headspace test.
Fig. 6:
The degradation of the reference substance
aniline in standard test methods. The graph
shows the average value curve of a standard
test determined from individual tests.

4.3 Compliance with validation criteria in the degradation of the

reference substance
The degradation results of all tests of aniline have been regularly
recorded and statistically evaluated in the laboratory for ecological
tests for some years. In addition to the measuring data in the blank
value preparations, the shape of the degradation graphs and kinetic
quantities derived from it are the quality criteria, which form the basis
of the decision on the validity of a successfully running test. Thus, test
results are only accepted if the reference substance was degraded after
14 days to over 60 % in the manometric respiration test, CO2
development test and CO2 headspace test or to at least 70 % in the Die
Away test. In the case of aniline, this validation criterion is not only
achieved in all standard test methods (Fig. 6), but also fulfilled
without any problem in the performance of the manometric respiration
test in the OxiTop Control test system (Fig. 7).
4.4 The degradation of the reference substance aniline in the

comparison of the Sapromat / OxiTop Control test systems
The aniline degradation graphs of the two types of respirometer differ
apparently only in the level of the rate of degradation in the
exponential phase (Fig. 7): With approx. 15 % degradation per day,
the maximum degradation rate in the OxiTop Control test system is
approximately one and a half times as high as in the Sapromat with
approx. 9 % degradation per day. In the case of aniline, this leads to
the limit of 60 % degradation that is so important for the evaluation of
the easy biological degradability of a substance being reached two
days earlier in the OxiTop Control test system.
Fig. 7:
Degradation of the reference substance
aniline in the Sapromat and OxiTop Control
test systems. The figure shows the average
value graphs determined by the individual
tests in both types of respirometer (% BOD of
the ThOD standard deviation).
Only degradation tests with a degree of
degradation of less than 100 % at the end of
the test were taken into account when
determining the OxiTop Control test system
average value graph.

4.5 Nitrification in the degradation of the reference substance

aniline in the OxiTop Control test system
Only five in ten respiration tests performed over a period of two years
were taken into account in the determination of the average
degradation graph of aniline in the OxiTop Control test system. Five
tests showed a degree of degradation at the end of the test of more
than 100 %. These samples showed the presence of nitrite and nitrate
at the end of the test that would suggest a participation of nitrificants
in the degradation of the nitrogen-containing compound. Figure 8
shows a typical example for the degradation of aniline in the OxiTop
Control test system. While nitrification started in one of three parallel
preparations after approx. 18 days and, therefore, the degradation
values at the end of the test lay at more than 100 %, the degradation
process ran in the two other vessels without the participation of
nitrificants.
4.6 Nitrification in the degradation of the reference substance

aniline in standard test methods
It was conspicuous in this evaluation that the participation of
nitrificants in the degradation of aniline in the two types of
respirometer occurred with varying frequency: While nitrification was
observed in approximately 50 % of the tests in the OxiTop Control
test system, this component turned out to be distinctly lower in the
Sapromat at approx. 7 %.
Fig. 8:
Nitrification in the degradation of the
reference substance aniline in the OxiTop
Control test system
In the end, the reason for this discrepancy could not be explained.
However, the following observation was made in an analysis of the
frequency of nitrification in the aniline degradation in different
standard test procedures: The nitrification of aniline is not only
restricted to the manometric respiration test in the OxiTop Control test
10

system, but occurred in practically all of the other analyzed standard

tests with the exception of the Sapromats (Tab. 2).
Checking this phenomenon revealed that nitrite and nitrate
concentrations were measured at the end of the trial for the blank
value and reference substance preparations of the different tests, from
which the average nitrogen concentration was calculated from the
nitrite and nitrate nitrogen (Tab. 2).
Tab. 2: Total nitrogen concentration of nitrite and nitrate nitrogen

(mg/l) in the degradation of aniline in blank value and test
substance preparations using different standard test
methods after a trial period of 28 days
Standard test methods No. Blank value Test substance
(n) Total N Total N
(NO2-N and NO3-N) (NO2-N and NO3-N)
(mg/l) (mg/l)
Manometric respiration test:
- Sapromat 9 2.0 0.4
- OxiTop Control test system 5 2.7 6.9
CO2 development test 12 1.8 2.9
CO2 headspace test 1 1.4 5.2
Die Away test 11 2.6 5.3
4.7 The degradation of aniline in the presence of the nitrification

inhibitor, allyl thiourea
The nitrification of aniline occurred spontaneously and proved to be
uncontrollable. Often, only individual parallel preparations were
affected, seldom all of them. Also, the shape of the graph and the final
degree of degradation in the plateau phase of the aniline preparations
of a test often deviated heavily from one another because the
nitrification started at different points of time or proved to be of
varying intensity.
The resulting problems in the interpretation of the trial results could
be avoided by the addition of 10 mg/l allyl thiourea to blank value and
test substance preparations of the OxiTop Control test system. Allyl
thiourea causes a complete inhibition of the nitrification of aniline
(Fig. 9). This test was also performed in the Sapromat with and
without the nitrification inhibitor for comparison purposes, and also
showed that allyl thiourea does in fact inhibit the nitrificants, but does
not otherwise affect the degradation potential of the organisms present
in the activated sludge: The degradation graph of a test performed in
the presence of allyl thiourea practically agreed with the graph of a
test without inhibitor that was assessed in the same test system and
was not nitrificated (Fig. 9).
11

Fig. 9:
aniline in the presence of 10 mg/l allyl
thiourea in the comparison of the Sapromat /
OxiTop Control test systems
4.8 The degradation of the reference substance benzoic acid in

the comparison of the Sapromat / OxiTop Control test
systems
According to the OECD test regulations, benzoic acid can also be used
as an alternative to aniline as the reference substance of standard tests.
In the two types of respirometer, benzoic acid was already degraded to
more than 60 % within a week and, as a result, fulfilled an important
validation criterion (see 4.3) for the degradation of a reference
substance (Fig. 10).
Fig. 10:
benzoic acid in the comparison of the
12

4.9 Prospects the multi-component measuring system

In cooperation with the company WTW, a multi-component
measuring system was designed with automatic online recording of
the biochemical oxygen demand and the formation of carbon dioxide
(Fig. 11). If required, the two measurement parameters could be
complemented by DOC measurements in manually drawn samples.
The new test apparatus is suitable for the checking of both soluble as
well as sparingly soluble and volatile substances.
The approach of the multi-component measuring system represents a
combination of the OxiTop Control test system (Fig. 2) and modified
Sturm test with integrated online CO2 measurement (Fig. 12), which
also represented an equally new test apparatus that resulted from a
cooperation between BASF and the company WTW and that enables
largely automatic data recording and evaluation in the CO2
development test.
Fig.11: Fig.12:
The multi-component measuring system - a Modified Sturm test with integrated online
test apparatus for performing substance CO2 measurement, a test apparatus for the
analyses with automatic recording of the CO2 CO2 development test
development and of the BOD consumption as
1 = 2.5 l culture vessel; 2 = absorption
well as an off line determination of the DOC container with gas distribution capillaries; 3 =
elimination conductivity electrode; 4 = air inlet; 5 = air
1 = 2.5 l culture vessel; 2 = absorption outlet
container;
3 = conductivity electrode; 4 = OxiTop
Control probe; 5 = sampling point
13

As in the OxiTop Control test system, the carbon dioxide in the

multi-component measuring system is created by the oxidation of a
carbon compound and is absorbed by a lye that is located in a
collecting container integrated in the closed culture vessel. An
OxiTop Control probe registers the resulting negative pressure and a
BOD value is derived from it. A conductivity probe immersed in the
absorber solution records the conductivity whose value decreases with
the absorption of the carbon dioxide. The amount of carbon dioxide
formed is determined from the conductivity values by means of a
calibration line and the biological degradation is calculated by
comparing the amount of carbon dioxide with a reference value. The
system also has a sampling point where the sample can be drawn from
above the culture liquid in order, e.g. to determine the DOC
elimination or the development of the biomass (Fig. 11).
The advantages of the multi-component measuring system are that
differences, e.g. as were observed in the degradation of aniline in the
Die Away test and manometric respiration test (Fig. 6), can be
detected and evaluated in a single test system. The participation of
nitrificants in the degradation of aniline (Fig. 9) can be determined
just as easily by means of the difference between the CO2
development and BOD graph. It can then be balanced so that aniline
can also be used as a reference substance when performing the
manometric respiration test in the OxiTop Control test system
without the use of a nitrification inhibitor as in all other standard tests.
The WTW company has registered patent rights for the multi-
component measuring system and for the modified Sturm test with
integrated on line CO2 measurement. In all probability the system will
be introduced onto the market in 2001 after concluding the
development activities in the laboratory for ecological tests of the
BASF.
5. The manometric respiration test in the presence

of different substance concentrations
If, for a degradation test using the manometric respiration test, the
degree of degradation in an inhibition preparation that contains the
same amounts of test substance and reference substance is less than 25
%, after a trial period of 14 days, then a toxic effect of the substance
on the activated sludge can be assumed. According to the OECD
validity criteria, the test should be repeated for these cases with a
lower test substance concentration or with a higher inoculum density
that must not, however, exceed 30 mg/l dry weight.
Figure 13 shows that the test substance concentration can be halved in
both types of respirometer without the degradation graph deviating
significantly from a test with a standard concentration (100 mg/l). The
validity criteria for the inhibition preparation are also still fulfilled at a
substance concentration of 25 mg/l.
14

Fig. 13:
Performance of the manometric respiration
test in the presence of different aniline
concentrations
6. Biological degradability of substances in the comparison of the

The biological degradability of substances that differ in molecular size
and structure was tested in both the Sapromat and the OxiTop
Control systems. The tests were usually started at the same time. The
aim of the tests was to establish to what extent the degradation graphs
and the evaluation of the biological degradability carried out on the
basis of the OECD criteria agree.
For biologically readily degradable substances, the degradation graphs
agreed quite well as a rule so that the evaluation of the biological
degradability turned out to be consistent (Tab. 3; Fig. 10, Fig. 4 c, Fig.
14 a, Fig. 14 c-e). The slight differences lay in a range that was
tolerable for biological systems.
In the first Sapromat test, BASF 980776 (Fig. 14 c) showed itself to
be poorly biologically degradable whereas in the corresponding
OxiTop Control test system trial, however, it was found to be easily
biologically degradable. After the stirrer normally used in the
Sapromat was replaced by a smaller version, the easy biological
degradability was also confirmed by repeating BASF 980776.
In BASF 980708 (Fig. 14 b), BASF 990020 (Fig. 14 g) and BASF
980785 (Fig. 14 h), the different degradation rates of the substances in
the two test systems also led finally to evaluations of the biological
degradability (Tab. 13) that deviated from one another. Whether the
differences as in the case of BASF 980776 was connected with the
15

prevailing high-energy entry in the two systems or were caused by

other effects could not be resolved any further.
Significant advantages as regards the rate of degradation or the level
of the degree of degradation in the plateau phase in favor of one of the
two systems were not observed in the simultaneously performed tests.
7. Summary
Extensive tests were carried out in the laboratory for ecological tests
of the BASF to find out whether the OxiTop Control test system is
suitable for performing biological degradation tests in the manometric
respiration test and, as a result, whether a further type of respirometer
is available for substance tests in addition to the Sapromat.
In particular, attention was paid to compliance with the validity
criteria given in the OECD Guideline:
In fact, after 28 days, the BOD values of the blank value
preparations exceeded the range of 20 30 mg O2/l specified in
the guideline by approx. 35 mg O2/l. However, the critical value
of 60 mg O2/l was not exceeded.
Parallel preparations may only deviate from one another by a
maximum of 20 % at either the end of the test, in the plateau
phase or at the end of the 10-day window. Also this validity
criterion was usually met. Frequently, the degree of deviation as
also in other standard test methods increased in less readily
degradable compounds.
16

Fig. 14 a-i: Degradation of substances in the comparison of the Sapromat / OxiTop Control test systems
17

Tab. 3: Evaluation of the biological degradability of substances in

the comparison of the Sapromat / OxiTop Control test
systems
Substance Evaluation of the biological degradability

Easily Biologically Partially/moder Badly
biologically ately degradable
degradable degradable degradable
Aniline OxiTop
Sapromat
Benzoic acid OxiTop
Sapromat
Diethylene glycol OxiTop
Sapromat
Isopropyl amine OxiTop
Sapromat
Isopropyl amine OxiTop
(+ATU: 10 mg/l) Sapromat
BASF 980708 Sapromat OxiTop
BASF 980776 OxiTop Sapromat
Sapromat (2) (1)
BASF 980803 OxiTop
Sapromat
BASF 990020 Sapromat OxiTop
BASF 980785 OxiTop Sapromat
BASF 980809 OxiTop
Sapromat
(1) Simultaneous test performance in the Sapromat and OxiTop Control test systems;
(2) Test repetition in the Sapromat
The reference substances, aniline and benzoic acid, were

biologically degraded to more than 60 % within 14 days. This also
complied with the validation criterion formulated regarding the
rate of degradation of the reference substance.
Aniline was frequently degraded in the OxiTop Control test system
with participation of nitrificants, which could be proved by means of
nitrite and nitrate analyses. The nitrification of aniline was not only
restricted to the manometric respiration test in the OxiTop Control
test system, but also occurred in practically all other standard test
procedures such as the CO2 development test, the CO2 headspace test,
the Die Away test and the Zahn Wellens test with the exception of the
Sapromats.
18

The nitrification of aniline could be suppressed by adding the

nitrification inhibitor allyl thiourea to the medium. In these cases, the
shape of the degradation graph agreed with the degradation behavior
of tests that showed no nitrification.
The test performance in the multi-component measuring system
provided a further possibility in avoiding the problems connected with
the nitrification of nitrogen-containing substances. The multi-
component measuring system provides a test apparatus that can
simultaneously trace the biochemical oxygen demand and CO2
development. As a result, the nitrification of nitrogen-containing
compounds can be determined in a single test system by means of the
differences between the CO2 development and BOD graph.
Degradation tests that were carried out at the same time in the
Sapromat and OxiTop Control test system proved that the
degradation graphs obtained from tests with biologically easily
degradable compounds usually agree to a large extent. However, it
was possible for the differences between the two types of respirometer
in the test of less easily degradable compounds to increase. In isolated
cases, this led to a different evaluation of the biological degradability
on the basis of the OECD criteria.
No clear advantages in favor of one of the two respirometers with
regard to the rate of degradation in degradation tests or the level of the
amount of degradation in the plateau phase were observed.
From the point of view of the laboratory for ecological tests, the
OxiTop Control test system provides a practical replacement for the
Sapromat and will be used increasingly in the future, not least due to
its simple operation and high reliability in the routine operation in
degradation tests.
19
Further development of the biological degradation test procedure...
Further development of the biological

degradation test procedure considering
respirometry
Prof. Dr. Uwe Strotmann, Fachhochschule Gelsenkirchen
1. Principles of aerobic biological degradation

Aerobic biological degradation processes play a decisive role in
natural and artificial ecosystems in the degradation of organic
substances. The principle that forms the basis of the degradation
processes is quite simple and universally realized by microorganisms.
Organic substances are assimilated by the cells of the bacteria and
transformed within the cells of the bacteria. In an aerobic
environment, this transformation is performed by an oxidation process
using molecular oxygen that is catalyzed by bacterial enzymes in
several stages. The object of this oxidation for the bacteria is that the
oxidation processes produce energy in the form of ATP. These
oxidation processes include the tricarboxylic acid cycle and the
breathing chain amongst other things. This process chain is also often
described as catabolic metabolism. The final products of the complete
aerobic degradation are carbon dioxide, water and simple inorganic
salts. On the other hand, intermediate products of the degradation can
also be used for the synthesis of new cell material. This includes the
organic substances that are assimilated by the cells and not completely
oxidized, but transformed into intermediate products that can then be
further transformed in the cells into in-cell material. This branch of
metabolism is described as anabolic metabolism. The bacteria that
perform these two process chains are following two objectives that are
essential for their survival:
a) The production of energy
b) Growth and reproduction
As a result, it would appear reasonable that the organic material that is
added to an aerobic biological degradation, should not be completely
oxidized to carbon dioxide, but should also be used for growth and
reproductive processes (Figure 1). Therefore, complete biological
degradation cannot be expected in biological degradation tests either
and is also seldom observed in practice. In this respect, the
heterotrophic productivity coefficient is a coefficient of measure that
specifies how much organic material is used for growth and
reproduction and how much is used for energy production.
1st Symposium on Biological degradability.. 26th September, 2000 Theme 3: Prof. Dr. Uwe Strotmann
Fig. 1.
Catabolic and anabolic metabolism in
bacteria
2. The OECD 301 test systems

The OECD differentiates between three principle test systems:
a) Test systems for ready biodegradability
b) Test systems for inherent biodegradability
c) Simulation test systems
These three principle blocks reflect the test philosophy of the OECD
quite accurately. The test systems for ready biodegradability are
very stringent test systems in which the conditions for the aerobic
degradation process are less than optimum. If the substances in this
test are degraded to a large extent, it can be assumed that degradation
in natural and artificial ecosystems is certain to occur. In the test
systems for inherent biodegradability, the level of stringency is
already lower; the conditions for degradation are already considerably
better here. In particular, the ratio of biomass to organic test substance
is considerably higher here than in the test systems for ready
biodegradability. The simulation test systems represent very special
environmental situations and the degradation is tested under these
special conditions. Naturally, the validity of this test is then also
restricted as a result of the special conditions.
For the test of ready biodegradability, there a total of 6 test systems
that exhibit a certain redundancy with regard to the measurement
parameters:
301 A: DOC die away test
301 B: CO2 evolution test
301 C: MITI (I) test
301 D: Closed bottle test
301 E: Modified OECD screening test
301 F: Manometric respirometry test
The parameters that are each determined individually in the test
systems are as follows:
Decrease in dissolved organic carbon (DOC): 301 A, E

CO2 production: 301 B
Oxygen consumption: 301 C, D, F
On closer inspection of the parameters, it becomes apparent that the

oxygen consumption alone is the most important measurement
parameter in three of the test systems. This underlines the significance
of respirometry. As an example of the test conditions, the conditions
for the OECD 301 F test are described below:
Conc. of the test substance: 100 mg/l ThOD
(theoretical O2 demand)
Temperature: 22C 1C
Inoculum conc. 30 mg/l TS (dry substance)
Test period: up to 28 days
pH value: 7.4 0.2
3. Development of a multi-component test system

To cut down the wide variety of test systems and also to make the
individual test systems more reliable by the monitoring of various
parameters, the obvious thing to do would be to combine several
parameters in a single test system (Fig. 2). The following
combinations would be possible:
a) Combination of DOC elimination and CO2 production
b) Combination of O2 consumption and CO2 production
c) Combination of DOC elimination and O2 consumption
d) Combination of DOC elimination, O2 consumption and
CO2 production
Fig. 2.
Illustration of the measuring apparatus in a
multi-component test system
Individually, these combinations have already been realized; further

combinations are being developed at the moment. This indicates that a
combination of these parameters makes sense in practice. The decisive
advantages of such a multi-component test system would be:
a) Several parameters provide higher reliability in the test evaluation
b) Kinetic parameters can be determined more easily
c) Mass balances can be set up
d) Lag phases can be determined more reliably
e) Better comparability of results
4. The determination of kinetic data from degradation graphs

The following parameters have been determined in OECD 301 test
systems so far:
a) Final degree of degradation
b) Lag phase
c) Compliance with the 10-day window
There have been some important developments in the field of

degradation kinetics over the last few years; a huge number of models
have been set up which are able to model the degradation graphs
mathematically.
Two well-chosen models are briefly described in the following:
1. Pseudo first-order kinetic
2. Modified logistic modeling
The pseudo first-order kinetic takes the data from the exponential
phase of the degradation graph and analyzes them. Data from the lag
phase or stationary phase are unsuitable and would distort the result.
The functions that are used for the modeling are as follows:
k t
Srem = Sinit e 1
ln(S init / Srem )
k1 = t
where Sinit is the initial substrate concentration, Srem is the remaining

substrate concentration, t is the time (in days) and k1 is a
characteristic degradation constant (first-order constant). The variable

that is to be determined by this modeling is the constant k1, a measure
of the rate of the degradation. The half-life t1/2 can also be easily
determined from these constants and is a very descriptive variable.
The precision of the modeling can be increased by submitting as many
data as possible from the exponential phase.
The modified logistic modeling uses not only data from the
exponential growth phase, but all the data that are available. The
entire degradation graph is then modeled. The following function is
used for the modeling:
S rem 1
S init = L (B + A e kt ) F
where L, A , B and F are four variable parameters that are adapted to
the measurement data during the modeling process and can also vary
over a wide range. These parameters do not have any direct practical
significance. Again the purpose of the modeling is the determination
of the degradation constant k. This form of the modeling requires an
adequate number of measurement data over the entire range of the
degradation graph in order to achieve a satisfactory model. After the
modeling, an optimized function is available and corresponding
degradation data can easily be calculated from this function. Examples
of both of these types of modeling are given in the following diagrams
(Fig. 3 to Fig. 6).
Fig. 3. Modified logistic plot of the degradation Fig. 4. Pseudo first-order plot of the
of diethylene glycol degradation of diethylene glycol
Fig. 5. Modified logistic plot of the degradation of Fig. 6. Pseudo first-order plot of the degradation of
morpholine morpholine
5. On-line determination of the degradation product, carbon

dioxide
After the start of the actual degradation phase, carbon dioxide (CO2) is
produced continuously by the biological degradation process.
Consequently, it would be an advantage if a continuous CO2
measurement process were available. Such a measurement could be
realized if the resulting CO2 is precipitated by a suitable adsorbent and
this precipitation could be quantified. In practice, the CO2 can be
conducted through a KOH solution (c= 25 mM) where it is
precipitated as K2CO3. The precipitation causes the ion intensity in the
KOH solution to decrease which can be quantified using a
conductivity electrode. After appropriate calibration, an online CO2
measurement can be established in this way (Figure 7).
Fig. 7.
Calibration of the online CO2 determination
6. Multi-component systems in practical applications

Some degradation graphs for multi-component systems are shown
below that were also correspondingly modeled.
6.1 Degradation of morpholine

The degradation of morpholine was traced by measuring the oxygen
consumption and carbon elimination (DOC elimination) (Fig. 8). In
this case, it was observed that the lag phase up to the start of the actual
degradation phase was approx. 16 days. The graphs of the DOC
elimination and the oxygen consumption were almost identical. The
final degree of degradation lay much higher than 80 %. The marked
lag phase of 16 days was easily reproducible in several trials and was
subject to only slight fluctuations. Lag phases of similar length were
also observed by other authors.
Fig. 8:
Degradation kinetics of morpholine
6.2 Degradation of diethylene glycol

The degradation of diethylene glycol was traced by measuring the
CO2 production and DOC elimination. Here, as expected, the graph of
the CO2 production was time-shifted with regard to the graph of the
DOC elimination (Figure 9). Also, the degree of degradation that was
calculated from the DOC elimination was higher than the degree of
degradation that was calculated from the CO2 production. The
explanation for this has already been described above and is linked
with the heterotrophic productivity coefficients.
Fig. 9.
Degradation kinetics of diethylene glycol
6.3 Degradation of aniline

The degradation of aniline was measured with the aid of three
parameters:
DOC elimination
Oxygen consumption
CO2 production
The degradation behavior is shown in the following diagram (Fig. 10).
The degradation graphs behaved as expected here. Firstly, after a lag
phase of approx. 3 days, DOC elimination began and proceeded very
rapidly. Shortly afterwards the uptake of oxygen and the CO2
production took place and resulted in largely comparable graphs that
confirmed one another. Also in this case, the degree of degradation
that was calculated from the DOC elimination was higher than the
degree of degradation resulting from the O2 consumption and the
degree of degradation resulting from the CO2 production.
Fig. 10:
Degradation kinetics of aniline
6.4 Kinetic data from different test systems

The following table (Tab. 1) provides a summary of the kinetic data
for the degradation of various substances. As can be seen from the
table, the k1 constants for a pseudo first-order modeling can be
scattered over quite a wide range. However, typical values lie within
the range 0.10.5 (unit: per day) which correspond to half-lives of
1.38 to 6.93 days. Exceptions are sometimes observed in the DOC
elimination process as this can occur very rapidly under certain
conditions (e.g. aniline).
Tab. 1. Summarized presentation of kinetic data of the degradation

of different organic substances. The k1 values are based on
analyses of pseudo first-order plots.
7. Summary
There are a series of OECD 301 degradation test systems that are
based on the parameters of DOC elimination, oxygen consumption
and on the production of carbon dioxide. These three parameters are
used for the assessment of the degradation behavior. By combining
the parameters into a single test system, the OECD 301 test strategy
can be standardized and also simplified. Moreover, by monitoring
several degradation parameters, the assessment of the results become
considerably more reliable and the comparability of the results is
improved. A further significant step towards the improved assessment
of results from degradation tests is the mathematical modeling of the
measurement parameters. Suitable models have already been
developed; Examples of this are the modified logistic plot and the
pseudo first-order plot.
10

Determination of the biodegradability of wastewater using the OxiTop Control
Determination of the biological

degradability of wastewater using the
OxiTop Control System
Martina Defrain, RWTH Aachen
1. Introduction
The spectrum of wastewater analysis ranges from the often time-
consuming DIN analysis through the simple and economical short-
time test analysis up to largely automated process analysis.
Sum parameters such as the biochemical oxygen demand (BODn),
the chemical oxygen demand (COD), the total organic carbon
(TOC) and the filterable solids play an important role in the
assessment of the load situation of a wastewater treatment plant. Only
via the determination of such parameters is it possible to assess the
purification capacity of a wastewater treatment plant with justifiable
expense.
Even in the optimum operation of a wastewater treatment plant, the
organic loads in the effluent of the biological stage determined via
these parameters can be so high that the effluent values are exceeded.
In phase separation that is functioning well, a large part of the residual
organic pollutant loading in the effluent is caused by dissolved,
biologically inert compounds. A more extensive elimination is only
possible via special treatment processes where the associated financial
and operating costs are considerable. Consequently, it is important to
create conditions to identify the entry path of biologically badly
degradable or inert organic substances into wastewater purification
plants.
For this, it is necessary to develop a method for the determination of
the biological degradability of wastewater components. Thus, if
necessary, influence can be brought to bear on the indirect passings in,
on the management of wash water containing sludge and on the
operating processes in the individual stages of the method (regulation
of the oxygen supply, change in the dry substance content, etc.).
Above all, an instrument should be placed in the hands of the
operators of smaller plants that makes operation safer and that enables
operating optimization to be carried out.
1st Symposium on Biological degradability.. 26th September, 2000 Theme 5: Martina Defrain

2. Degradability of wastewater components

For the determination of the degradability of both the dissolved and
particulate constituents in wastewater, long-term degradation trials can
be performed using wastewater samples.
Figure 1:
Long-term degradation trials using
wastewater samples (diagrammatic sketch)
/2/
Preparations for long-term degradation trials

Wastewater ATU 1 ml activated sludge
Preparation 1 Preparation 2 Preparation 3 Preparation 4
COD g
COD f
Analysis after Analysis after

0 days 5 days
consumption consumption Analysis after
21 days Analysis after
consumption 28 days
consumption
COD g
COD f
COD g
COD f COD g
COD f
The trial is carried out under the following conditions:

The degradation trials are carried out at a constant temperature of
20 C.
For each individual wastewater, at least 4 preparations are
produced (in Sapromats, each with a volume of 250 ml).
If necessary, 1 ml activated sludge is added to the preparations.
The trials are carried out with the addition of ATU to inhibit
nitrification.

The preparations are analyzed at the following times:

Preparation 0, before the addition of activated sludge
Preparation 1, immediately after the addition of activated sludge
Preparation 2, after 5 days
The following analytical parameters are determined from the original
sample and preparations 0 to 4.
COD g chemical oxygen demand of the whole
homogenized sample
COD f chemical oxygen demand of the filtered sample
The TOC content and the DOC (dissolved organic carbon) content can
also be determined in the same way.
The trial evaluation is carried out under the following conditions:

The particulate quota (CODp, POC) of the components is
determined mathematically from the difference of the
concentrations in the homogenized and filtered samples.
The values of the original sample (preparation 0) before the
addition of activated sludge are taken as the initial concentrations
of the dissolved and particulate wastewater components.
Dissolved biologically inert substances from the degradation of the
heterotrophic biomass after a 28-day period of consumption are
quantitatively ignored.
For the assessment of the degradability of wastewater components, the

following classification is used:
Readily biologically degradable:
Wastewater components that degrade after 5 days are defined as
readily biologically degradable.
Fairly biologically degradable:
Wastewater components that degrade between 5 and 21 days are
defined as fairly biologically degradable.
Moderately biologically degradable:
Wastewater components that degrade between 21 and 28 days are
defined as moderately biologically degradable.
Maximally inert:
The remaining dissolved wastewater components represent the
maximum value of inert CODf-causing compounds (or DOC

content). For the biologically inert particulate wastewater

components (CODp, POC), only the limits can be indicated by the
increase and decrease of the biomass. The particulate components
that remain after a 28-day consumption period are made up of the
heterotrophic biomass, the particulate inert organic degradation
products from the biomass and the inert CODp-causing compounds
(or the POC content) of the original sample. These remaining
particulate wastewater components represent the maximum value
of inert CODp-causing compounds (or TOC content).
3. Trials using the OxiTop Control system

The OxiTop Control manometric measuring system of the WTW
company in Weilheim is used in many refinery laboratories of
wastewater treatment plant operators for the determination of BOD5.
Since the method introduced in chapter 2 for the determination of
readily and poory biologically degradable organic wastewater
components was developed especially for the operation of wastewater
treatment plants, it was obvious that analyses should be carried out to
check the suitability of the OxiTop control system for this
determination procedure.
The trials were carried out in graduated flasks with a nominal volume
of approx. 510 ml in which the enlarged sleeves are suspended. The
oxidation of the wastewater components by the bacteria creates CO2,
which is fixed in the sleeves by NaOH pellets. Pretrials clearly
showed that long-term measurements with the small sleeves used in
the determination of BOD5 led to an overflow of the liquid formed by
the reaction of the CO2 with the NaOH pellets.
A total of 24 graduated flasks were prepared for the trial series
described here. Each of these was filled with the same amount of
wastewater. The samples were mixed with 2 ml ATU to inhibit
nitrification. Three NaOH pellets were inserted in the sleeves, the
measuring heads were screwed on and the bottles were placed in two
thermostat boxes (20C).
The preparation was carried out with a sample volume of 97 ml
original wastewater from the inflow to the activation tank of a single-
stage wastewater treatment plant with a design capacity of around
460,000 population equivalent.
Before pouring in the samples, the content of the bottles was measured
in liters. The exact volumes were entered in the associated controller
and, thus, could be automatically incorporated in the calculations of
the measured values. No activated sludge was added to the samples.
As the samples only concerned the mechanically pretreated
wastewater of a municipal wastewater treatment plant, sufficient
bacteria were present to ensure degradation processes.
The nutrient ratio in the samples at the beginning of the trial were in
an optimum range (C:N:P = 100:23:3) for the growth of the biomass.

The remaining headspace in the graduated flask should be as large as

possible in order to ensure sufficient oxygen to last for the entire
consumption period. If a sample volume of 97 ml is used, a headspace
of approx. 413 ml remains. Provided that the analyses are carried out
at an air pressure of 950 mbar and a temperature of 20C, around
100 mg O2 is available. COD analyses of the wastewater result in
values of between 500 and 600 mg/l COD. This corresponds to an
oxygen demand of around 50 to 60 mg for an initial volume of 97 ml.
Thus, even with complete oxidation of the wastewater components,
sufficient oxygen was available. Measurements were carried out over
a total period of 28 days. In each case, two COD analyses were carried
out after 0, 3, 5, 17, 18, 19, 20, 21, 22, 23, 24 and 28 days.
A measured value of the consumption was recorded every 112
minutes. Figure 2 shows the consumption graphs of the 24 samples. In
this case, fluctuations of up to 50 mg/l O2 occurred in the consumption
curve. Scattering in the range of up to 20% were also determined in
long-term measurements with the OxiTop measuring system by
CONZELMANN/BEITLICH /1/.
Figure 2:
Consumption graphs of the individual
preparations (OxiTop)
The COD of the original homogenized sample (CODg) lays at 552

mg/l.

The following diagram shows the compilation of all the results of the
COD analyses. Figure 3:
Results of the COD analyses
[mg/l]
600
500
CSBg
400
CSBf
CSBp
300
200
100
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Days
The dissolved wastewater components (CODf) are essentially

degraded during the first five days. This is connected with an increase
in the biomass of the sample, clearly identifiable on the third day,
where the particulate COD lies above the original CODp. Up to the
18th day of consumption, a continuous degradation of the particulate
wastewater components occurs. The dissolved COD remains almost
constant over this time period. On the 19th day of consumption, an
increase of the COD-causing wastewater components occurs that can
only be explained by biochemical processes.
Extensive analysis of the theoretical and actual COD values measured
by means of the DIN method resulted in considerable deviations
depending on the wastewater component /3/. In this case, the presence
of higher molecular substances with high theoretical but low
measurable COD could be possible. Through the formation of
metabolites, the proportion of measurable COD could have been
increased which would explain an increase in the COD concentration.
At the same time as the degradation of dissolved COD-causing
components an increase of the particulate COD and the oxygen
consumption occurs again on the 19th day determined by the BOD.
Between the 20th and 28th day, the COD of both the dissolved and of
the particulate constituents levels out to an almost constant value.

According to tables 1 and 2, the constituents of the readily, fairly,

moderately degradable and maximally inert COD-causing compounds
are determined.
Table 1: COD and BODn concentrations of the individual

preparations
Analysis results
Preparation Preparation Preparation Preparation Preparation

0 1 2 3 4
Consumption period 0 days 5 days 21 days 28 days
Parameter Analysis
CODg [mg/l] 552 only with 217 93 88.5
the addition
of activated
sludge
CODf [mg/l] 417 49 34 30
CODp [mg/l] (CODp = CODg - CODf) 135 168 59 58.5
BODn [mg/l] / 390 471 476
Table 2: Degradability of the COD-causing compounds
Fractionation acc. to biological degradability
Readily Fairly Moderately Maximal

degradable degradable degradable inert
Values from: Preparation 0 - Preparation 2 - Preparation 3 - Preparation 4
preparation 2 preparation 3 preparation 4
CODf [mg/l] 368 15 4 30

CODp [mg/l]: (-33) 76 0.5 58.5
(Preparation 0 -
preparation 3)
Due to the high increase in the biomass in the first days, no readily
biologically degradable constituent of the particulate COD-causing
components can be determined. After 5 days of consumption, the
COD concentration is higher than that of the initial concentration. For

this reason, the original COD (preparation 0) is selected for the

determination of the moderately biologically degradable components.
The following figure graphically depicts the individual fractions.
Figure 4:
Fractionation of the COD causing compounds
COD causing compounds

552 mg/l
CODp CODf
24% 76%
Fractioning according to biological degradability

Moderately
Maximall 1% Maximally inert
inert Fairly 7%
43% 4%
Fairly
57%
Moderately
(0.4%)
Readily
88%
The diagram clearly shows that the largest proportion of the dissolved
wastewater components was easily degradable. A maximum of 7% of
the dissolved wastewater components was determined to be inert. A
completely different picture is shown for the solid matter. Of the
COD causing components present in the form of solids in the original
sample, 43 % still remained after 28 days. This involves a
heterotrophic biomass created during the biological degradation, inert
particulate organic degradation products and inert particulate COD-
causing compounds from the original sample.
4. Prospects and summary

The OxiTop control system showed itself to be well suited for the
application purposes described in this publication. Since many parallel
preparations are necessary for the fractionation of the COD-causing
compounds in wastewater, a measuring system with simpler operation
is beneficial. Special temperature-adjustable boxes with an integrated

magnetic stirring system guarantee constant conditions for all

preparations during the analyses.
Referred to the BOD28, fluctuations with a maximum of 11.2 %
occurred in the consumption curve. In comparable long-term
measurements /2/ with a Sapromat, the fluctuations lay between 11
and 23%.
A further fractionation of the maximally inert COD-causing
components can be carried out. Measurements on the first and second
day of consumption enable the determination of the heterotrophic
biomass growth (measured as COD) through the degradation of the
dissolved COD-causing compounds in wastewater. If this entire
heterotrophic biomass is degraded during the consumption period of
28 days, particulate COD-causing compounds remain from this
biomass that can be calculated, e.g. via the SIMBA simulation pro-
gram for biological wastewater purification /4/. The inert substances
remaining from the degradation of the biomass (Xp) can be
determined for various COD values. From that, the Xp of the biomass
can then also be calculated from the degradation of the particulate
COD-causing compounds. If the values for Xp of the remaining
particulate COD-causing wastewater components are subtracted, the
inert particulate quota of the original sample remains. Appropriate
simulations and orientation aids for practical applications are being
developed at the moment and, in the future, will enable a rapid
assessment of the inert particulate COD-causing compounds present in
wastewater. /2/
5. Bibliography
/1/ Conzelmann, F.; Beitlich,R.:
Einfache Bestimmung der biologischen Abbaubarkeit nach
OECD 301F mit dem OxiTop
melliand Sonderdruck 10/1996, 700-702
/2/ Defrain, M:
Praxisrelevante Bestimmung und Bewertung biologisch leicht
und schwer abbaubarer Stoffe in kommunalem Abwasser
Dissertation in Vorbereitung, 2000
/3/ Janicke, W.:
Chemische Oxidierbarkeit organischer Wasserinhaltsstoffe
Dietrich Reimer Verlag, WaBoLu Berichte, Heft 1, 1983
/4/ NN:
SIMBA 3.4+ - Klranlagen Simulation unter Matlab/Simulink
Institut fr Automation und Kommunikation e.V., Magdeburg,
1999
OxiTop for the determination of the respiratory activity of soils and solids
Validation of the OxiTopmeasuring

system for the determination of
respiratory activity in soils and other
solids
Prof. Dr. Harald Platen, FH Gieen-Friedberg
Keywords
Soil respiration - Respiratory activity - Pressure measurement - Solid
matter - Soil - Oxygen consumption
Abbreviations
BOD: biochemical oxygen demand; OS: organic substance;
DS: dry substance
1. Introduction
1.1 The measurement of the respiratory activity in soils

For decades, biological activity has been used as a measurement
variable in the analysis of water in routine measurement procedures
(BOD, spontaneous oxygen consumption [1, 2]) to assess pollutant
effects. However, comparable procedures in soil analysis have
achieved no great significance up to now although they have been
recognized for a long time [3]. Among other factors, this lay in the
fact that there were no legal requirements for appropriate
measurements and that no measuring procedure was available that was
simple to use: they were either time consuming or demanding with
regard to instruments and, consequently, cost intensive (for an
overview, see [4]). Through the increasing demand in the sector of
residual pollution for biological soil development procedures and also
due to requests for treatment of biological refuse, an increasing
requirement arose for a simple biological measurement procedure for
solid matter and recommendations for measurement procedures were
laid down [5, 6]. After a new measuring system on the basis of the
measurement of a decrease in pressure (OxiTop, WTW, Weilheim,
FRG [7]) was available for the wastewater sector, procedures for the
measurement of solid matter were also developed [8, 9, 10, 11].
Within the framework of the introduction of the
Bundesbodenschutzgesetzes (Federal Soil Protection Bill) [12] and
subsequent implementation regulations, we will now discuss citing the
1st Symposium on Biological degradability.. 26th September, 2000 Theme 6: Prof. Dr. Harald Platen
determination of the respiratory activity of soils as the evaluation

criterion of the soil quality. A norm with the description of applicable
test procedures is being prepared [13] and, in more recent times, two
intra-laboratory tests on this topic have been carried out [23, 24]. Here
different preparations still appear in the evaluation of the test results
(e.g.: AT4: respiratory activity in 4 days, basal respiration, substrate-
induced respiration, lag-phase assessment, respiratorial activation quo-
tient, time to peak maximum). There still remains the necessity of
standardizing the specification of test results, which was referred to in
earlier works [25].
Equation 1 shows the reaction equation that is at the root of soil
respiration: organic substances are degraded to carbon dioxide and
water during the consumption of oxygen. This gives rise to two basic
options for measuring the respiratory activity: measuring the decrease
in oxygen or measuring the formation of carbon dioxide.
OS + x O2 y CO2 + z H2O [Equation 1]
At the same time, according to the organic substance used, the ratio of
oxygen consumption to the formation of carbon dioxide is dependent
on the average redox status of the carbon (see table 1). The soil itself
contains a complex mixture of organic substances whose degradation
causes the basal respiration. If oxygen consumption is considered as
the measurement variable, further biological processes
(nitrosofication, nitrification; equations 2 and 3) and also inorganic
reactions (in the presence of reduced mineral components; equation 4)
can lead to oxygen consumption. This fact must be taken into account
in order to interpret the results correctly. In the determination of the
formation of carbon dioxide as the measurement variable, it must be
noted that carbonate-rich soils can release carbon dioxide without this
correlating to any biological activity and that basic soils do not
completely release carbon dioxide. This must be noted in the
interpretation of respiratory quotients (ratio of oxygen uptake to
carbon dioxide formation). Moreover, this also explains why the
results of measurements of the decrease in oxygen and uptake of
carbon dioxide are not directly comparable with one another,
particularly not when the carbon source is not known or mixtures of
unknown compounds are present.
NH4+ + 1,5 O2 NO2- + H2O + 2 H+ [equation 2]
NO2- + 0,5 O2 NO3- [equation 3]
4 Fe2+ + O2 + 2 H2O 4 Fe3+ + 4 OH- [equation 4]
Table 1: Connection between the mean redox status of

organic compounds and the respiratory
quotients, calculated using the example of C1 compounds.
Redox Example of reaction equation Respiratory quotient

status
mmol O2/ mg O2/
mmol CO2 mg CO2
-4 CH4 + 2 O2 CO2 + 2 H2O 2.0 1.45
-2 CH3OH + 1.5 O2 CO2 + 2 H2O 1.5 1.09
0 HCHO + 1 O2 CO2 + H2O 1.0 0.73
+2 HCOOH + 0.5 O2 CO2 + H2O 0.5 0.36
+4 CO2 + ./. CO2 0.0 0.00
1.2 Limit monitoring

The introduction of legislative or administrative legal regulations for
monitoring the environment is usually linked with the definition of the
physical, chemical and biological parameters that must be monitored
using suitable procedures. Experience indicates that the same samples
can provide absolutely different measurement results when different
test procedures are used and, consequently, leads to the fact that only
defined procedures may be used for monitoring limits. In this case,
primarily the comparability of different test procedures plays an
important role: if a specific test procedure is stipulated for monitoring
a limit, alternative procedures may usually only be employed if the
equivalence of the test procedure has been proved. An important
prerequisite for the practicability of a test procedure is that it can be
validated.
1.3 Validation
The validation of a test procedure is understood to be a process that
includes its introduction and regular systematic assessment (to
validate or declare to be valid). The validation process can be mainly
depicted in 3 stages (table 2). Since, for each individual test or series
of tests, the validity of the procedure for the individual test situation
must be ascertained before the results are output, validation plays a
central role in the analysis. Validation data can be completely
determined in the in-house laboratory; standardized procedures can
also draw upon data that have been obtained in the course of the
standardization and have been published in connection with the norm.
Table 2: Stages of the validation of a test procedure
Validation status Extent of work / Steps

Phase 1 Basic calibration
a. Determination of the type of calibration function
b. Determination of the procedure data
c. Determination of the measuring precision (repeat measurement of a
standard)
d. Determination of the method precision (repeat measurement of a
sample )
e. Determination of the correctness / selectivity (recovery rate)
f. Assessment of the initial data for robustness of the procedure:
chronological, personnel, reagents and instrument-dependent stability
of the procedure.
Phase 2 Preparation of the routine phase
a. Creation of standard operating instructions
b. Training of the laboratory personnel
c. Continuation of the data collection for robustness and their continuous
evaluation :
Chronological stability
Effect of a change of personnel
Specification of data for quality control
Use of quality control cards
d. Specification of the procedure in quality problems
e. Specification of the data documentation
Phase 3 Routine phase
a. Regular use of and evaluation of control cards
b. Participation in laboratory comparison measurements (intra-laboratory
tests)
c. Carrying out of regular planned maintenance and calibration activities
d. Documentation
Within the scope of the task in hand, aspects are discussed here that
are of significance in the practical introduction and validation of the
OxiTop Control measuring system for the determination of soil
respiration as a routine procedure.
2. Material and methods
2.1 Determination of the respiratory activity:

manometric procedure using the OxiTop measuring system
20 g to 300 g of soil or solid sample with a water capacity that is
usually adjusted to half-maximum and sieved to 2 mm (wherever
possible and practical!) are weighed into in a preserving jar (550 mL,
1000 mL or 1500 mL volume). This is distributed in such a way that a
depression, into which a 50 mL beaker that contains 50 mL NaOH
(2.0 mol/L) is placed, is created in the middle. The edge of the
preserving jar and the seal of the lid-locking device (WTW, Weilheim,
FRG) are lubricated with some Vaseline and the vessel closed with 4
retaining clips. The OxiTop measured value recorder is screwed on
with a rubber seal and the preparation thermostatted for 3 hours in the
thermostat cabinet; measurement is then started with the OxiTop
OC110 controller. The measuring period is specified according to
requirements. Details are given in the operating manual [15]. If the
pressure drops by more than 100 mbar, the measuring vessel is
opened, ventilated and - if measuring is to be continued - the caustic
soda solution replaced and the measuring vessel closed again. For an
evaluation, see equation 6.
2.2 Determination of the respiratory activity : titrimetric

procedure (modified acc. to Isermeyer [3])
20 g to 100 g soil or solid sample with a water capacity that is usually
adjusted to half-maximum and sieved to 2 mm (wherever possible and
practical!) are weighed into a preserving jar (550 mL). This is
distributed in such a way that a depression, in which a 50 mL beaker
is placed that contains 50 mL NaOH (0,1 mol/L), is created in the
middle. The edge of the preserving jar and the lid is lubricated with
some Vaseline and the vessel closed using a rubber sealing ring with 4
retaining clips. Until the end of the measurement that complies with
the expected respiratory activity, the sample preparation remains in a
dark room at a constant temperature. After the incubation time has
expired, the vessel is opened and the NaOH is transferred into an
Erlenmeyer flask. 10.0 mL BaCl2 solution (0.5 mol/L) and some drops
of phenolphthalein solution (0.1% in 50% ethanol) are then added and
it is titrated with HCl (0.1 mol/L) until the color changes to colorless.
For the determination of the blank value, in each series of
measurements a preparation is produced as described above, although
without the use of soil. The evaluation is made using equation 5:
mgCO2 [Equation 5]
mCO2 = (VPr VBW ) 2,2
mL
mCO2: Mass of CO2 formed in [mg], VPr, VBW: Volume consumption

0.1 mol/L HCl in [mL] of sample (Pr) and blank value (BW).
Allowing for an adequate margin to the upper and lower measuring

range limit, the range of operation of the procedure lies between 1 mg
and 80 mg CO2 that may be formed by the soil used in the measuring
period in order to be utilizable. From a direct relationship between the
results obtained from equation 5 and the soil mass used (as TS) and
the incubation time, the respiratory activity rate is obtained in, e.g.
[mg CO2/(kgd)].
2.3 Determination of the dry substance content

An aliquot of the moist material (20 to 50 g) is placed in a porcelain
bowl and dried at 105C until it reaches a constant weight. The dry
substance quota of the material [16] is calculated from the mass
determinations of the tare as well as of the moist and dried material.
The material thus treated cannot be used for further tests.
2.4 Determination of the maximum water capacity

An aliquot of the material in the condition in which it should be used
for the determination of the respiratory activity (e.g. sieved) is placed
in a plastic tube (D x H: 5 cm x 6 cm), that is closed at the lower end
with a filter paper and gauze, to a height of approx. 5 cm. The
preparation is placed in a water bath so that the surface of the water is
approximately 1 cm above the surface of the material. After 2 hours,
the preparation is placed on a moist sand bath and covered with a
watch glass to prevent evaporation. After a further 2 to 3 hours,
approx. 40 g of the sample is removed using a spatula and the TS
content or water content is determined [17].
2.5 Adjustment to approx. the semi-maximum water capacity

The required water capacity of 50 to 60 % is adjusted by the addition
of deionized water or by slow drying the sample at room temperature.
3. Results and discussion
3.1 Fundamentals of the measurement procedure and calibration

The manometric procedure is based on the measurement of the
decrease of oxygen in the measuring system. The relationship shown
by equation 6 exists between the measurement result (amount of
oxygen consumed) and the decrease in pressure. Since the free gas
volume (Vfree) in a measuring preparation remains constant and the
temperature (T) during the measurement is also held constant, a linear
relation is inevitably revealed between the mass of oxygen consumed
(m) and the decrease in the pressure (p) since the mol mass (MR)
and the gas constant (R) are constants.
[Eq. 6]
Commonly, the general gas constant of R = 8.314 Jmol-1K-1 is used.

However, it must be pointed out that oxygen is not an ideal gas and, as
a result, has a slightly divergent specific gas constant R(O2) =
8.301 Jmol-1K-1 (at 0C and pressure of 1013 mbar) [14]. The use of
the general gas constant leads to a slightly positive proportional syste-
matic error of 0.16% that, however, should not have any significant
effect in practice.
3.2 Effect of the measurement environment on the measurement

Temperature is one of the environmental conditions that must be kept
absolutely constant. On the one hand, the biological activity changes if
the temperature changes but, on the other hand, this also has a direct
effect on the pressure measurement (equation 7).
T2
pT2 = pT 1 [Equation 7]
T1
With an initial measuring condition of 293 K and 1 bar pressure, a

temperature change of 1 K signifies a change in pressure of
3.5 mbar. With a resolution of the measuring instrument of 1 mbar,
this means that the temperature of a measuring preparation greatly
influences the measurement result and, consequently, must be kept
constant for good temperature regulation. However, the OxiTop
Control measuring system has a temperature measurement and
compensation function that mathematically compensates for smaller
temperature fluctuations; however, larger fluctuations must be avoided
due to the direct effect on the biological processes.
In practice, it has proved effective to preheat all vessels and reagents
to the subsequent measuring temperature that has to be maintained so
that, after the production of the measuring preparation, the time in
which pressure changes takes place as a result of temperature
adjustment remains as short as possible. After introduction into the
temperature-controlled incubator, the measured values taken in the
first three hours are usually ignored.
Not to be underestimated in this context is also the radiation of (sun)
light: in the case of solar radiation, light absorption by relatively dark
samples in the measuring preparation (e.g. soil) can very quickly lead
to a temperature increase inside the measuring system and, as a result,
to a falsification of the measured values.
On the other hand, one effect that can be ignored is the distribution
effect of oxygen between the gas phase and the aqueous environment.
Strictly speaking, in the determination of the oxygen consumption by
a pressure measurement, in addition to the oxygen present in the gas
phase, a specific quantity is still present dissolved in the aqueous
phase (attainment of equilibrium) that must be taken into account in
the quantitative determination of the rate of consumption. This plays
an important role in the manometric BOD determination as, here, the
amount of oxygen in solution lies in the percentage range due to the
comparatively large quantity of water. In the determination of the
respiratory activity in solids, the extreme case lies in the use of, e.g.
300 g soil with a 15% water content in a 550 mL measuring vessel and
a ratio of free headspace to water of 400 mL : 45 mL. As a result, the
amount of oxygen that is dissolved in the soil water varies in the range
of 0.5% and less. However, such a high soil quantity is only used if
extremely low respiratory activities are to be determined which is
relatively seldom. Since usually distinctly smaller weights of soil and,

consequently, significantly less water is introduced into the measuring
preparation, the amount of oxygen dissolved in the soil water is
negligibly small in comparison to the total amount of oxygen.
3.3 Determination limit and scope of work

The determination limit marks the limits of the lower scope of
operation of a test procedure. The test procedure under examination
here is dependent on four variables: the vessel volume, the weight of
solid matter, the measuring period and the resolution of the pressure
measurement. The resolution of the OxiTop measuring system is
1 mbar while 360 individual measured values are recorded for the
freely selectable measuring period.
Figure 1 shows the (idealized) curve of a respiratory activity
measurement. Within the scope of this study, the determination limit
is defined so that 10 mbar continuously proceeding pressure decrease
in the course of the considered measuring period must be verifiable.
Other users can define the determination limit quite differently.
However, the criteria must then be stated openly.
Figure 2 shows the dependence of the determination limit of the
weight on the solid sample and the vessel volume; the lowest
achievable determination limit lies in the use of approx. 300 g solid
matter in a 550 mL vessel and a measuring period of 10 days at
approx. 15 mg O2/[dkg TS]. The lower the selected weight of solid
matter, the larger the effect of non-homogeneities that can have a
negative effect on the representativeness of the partial sample.
Various series of measurements ([9], page 49-51; [18]) have proved
that weights of solid matter up to 300 g obviously have no great effect
on the rate of gas exchange if the filling is not too airtight.
The upper measuring range limit is determined by two parameters:
firstly, by the quantity of oxygen available and, secondly, through the
quantity of caustic soda solution that fixes the CO2 formed. Depending
on the respiratory activity as well as on the selection of the measuring
vessel and sample quantity, different time periods arise after which the
measurement must be terminated (table 3). With regard to the oxygen
supply, the measuring system is operated in such a way that the initial
oxygen content of 21% sinks to 11% at most (pressure decrease of
approx. 100 mbar) [19]. Experience has shown that this ensures the
oxygen content is not so low that it becomes a limiting factor to the
speed. We have not yet confirmed whether the oxygen supply can be
still further exhausted without affecting the measurement result.
Furthermore, it must be ensured that the caustic soda solution is not
too heavily neutralized for the absorption of the carbon dioxide. It
shows that at least a four-fold stoichiometric surplus should be present
so that no retardation of the absorption occurs here (at least 50 mL;
c = 2.0 mol/L) [20]. Alternatively, a sufficient quantity of soda lime
can also be used (e.g. 1.7 g in a 1000 mL vessel). The use of NaOH
pellets or conc. caustic soda solution (10 mol/L) must be avoided

because these are so hygroscopic that they cause the sample under
study to slowly dry out [20].
Figure 1:
Idealized representation of the decrease of
pressure by 10 mbar during the recording of
the 360 meas. points by the OxiTop Control
measuring system. The period over which the
measured data are recorded can be freely
selected. Both the period and the resolution
of the pressure measurement have an effect
on the determination limit.
Figure 2:
Dependence of the determination limit of the
respiratory activity determination using the
OxiTop Control measuring system on the
weight of soil and measuring vessel volume
over a measuring period of 10 days.
Table 3: Overview of the duration of a respiratory activity

measurement depending on the respiratory rate of a solid,
the weight of solid matter used and the selected vessel
volume
Respiration rate [mg O2/(dkg DS)]
Weight of Vessel 10 100 1000 2500
solid matter volume
[g DS] [mL]
Duration of the measurement [d]
20 1500 939.2 93.9 9.4 3.8
1000 610.8 61.1 6.1 2.4
550 315.3 31.5 3.2 1.3
100 1500 182.6 18.3 1.8 0.7

1000 116.9 11.7 1.2 0.5
550 57.8 5.8 0.6 0.2
200 1500 88.0 8.8 0.9 0.4

1000 55.2 5.5 0.6 0.2
550 25.6 2.6 0.3 0.1
300 1500 56.5 5.7 0.6 0.2

1000 34.6 3.5 0.4 0.1
550 14.9 1.5 0.2 <0.1
3.4 Measuring precision

The measuring precision in analytical procedures is usually
determined by measuring a standard several times (at least 6-fold). A
statement is then obtained on the scattering margin of the measured
values through the statistical evaluation of the measurement results
that can be mainly traced to the characteristics of the measuring
instrument. To keep test expenses low, the normal oxygen content of
the atmosphere can be selected as the standard here and, consequently,
e.g. the scattering of the measured values of the pressure change that
is caused by the absorption of atmospheric oxygen if specific
quantities of chemical substances [22] are compared. In this case, the
entire measuring system is checked (pressure sensor incl. density of
the measuring vessel); the comparison of the pressure changes in
different vessels also enables a statement on which of the vessels is
not airtight.
If the pressure sensor is OK and the vessels are airtight, a larger
scattering of the measured values can only be caused by larger
temperature differences or unequal light radiation (see section 3.2).
A further criterion for the characterization of the measuring precision
is the test of the chronological resolution of the measuring system;
10
particularly in the determination of respiratory rates, it must be

ensured that the time measurement functions correctly.
3.5 Method precision

The precision of a method is determined by measuring a real sample
several times. Conclusions can then be reached on the precision of
respective measurements from the statistical evaluation. In the
classical sense, the effect on the test result is recorded from the sample
matrix and, if necessary, from sample treatment steps. The respiratory
activity is a biological property that, even under relatively constant
boundary conditions, can be subject to large fluctuations. The concept
of method precision under these circumstances must be regarded
differently than in conventional chemical and biochemical analysis.
In the measurement of soil respiration, an appropriate test can look as
if a soil sample is very well homogenized and adjusted to the optimum
water content and then at least 6 identical test preparations are
produced. Homogeneous and relatively sandy soils produce quite
good identical results. However, if heavy fluctuations are obtained,
this can be due to several causes that need not necessarily be an
indicator of the bad quality of the measurement. The most probable
causes include inhomogeneities of the soil or solid sample, unequal
humidity conditions, etc. Moreover, errors can also occur as described
in section 3.4. Nevertheless, these are relatively simple to limit and, if
necessary, preclude.
3.6 Robustness
In the observation of the robustness of a test procedure, the effect of
heavily varying boundary conditions on the stability of the test results
is investigated. As a result, "boundary conditions" usually means a
heavily varying matrix. However, they can also include the effect of
varying sequences of operation or changing personnel.
As already explained in section 3.5, the determination of the
respiratory activity concerns a biological parameter for which a
relatively large fluctuation margin is not necessarily unusual. In
addition, solids and soils can frequently only be homogenized up to a
certain point so that larger scattering margins of the test results must
be accepted more often than is usually the case in chemical tests. The
effect of different working methods of different laboratory personals
can be minimized by unambiguous descriptions of the test procedure
in standard operating instructions, which should also incorporate
experiences that describe the repercussions any deviation from the
standard regulation has on the test result. Naturally, the longer an
operator works with a procedure, the more experience he gains that
can hardly be fully written down.
11
3.7 Monitoring of the test procedure using other independent

test procedures
A further procedure in the process of validation is the examination of
the OxiTop Control measuring system using other independent
measurement procedures. While doing so, attention must be paid to
the fact that this is only practical with procedures that go back to the
same measurement parameter (oxygen consumption); the
determination of the carbon dioxide formation does not necessarily
provide adequate results since different organic substances - and, as a
result, e.g. also the expected variety of substances in natural soils -
show different respiratorial quotients and other respiratory processes
(e.g. nitrification) have an effect on the ratio of the consumed oxygen
to the carbon dioxide formed.
The equivalence of the OxiTop Control measuring system with the
Sapromat was demonstrated repeatedly (e.g. [21]). For laboratories
that introduce the procedure into their laboratory, it is sufficient to
refer to such works. In addition, it is possible to determine the carbon
dioxide formation and, as a result, the respiratory quotients in the
same measuring preparation in a simple way. For this purpose,
attention must be paid to the precise adjustment of the concentration
of the absorption solution used (NaOH) so that meaningful results
about the titration are obtained, and to preparing a control preparation
without soil in order to determine the titration blank value. However, a
weakness of the titrimetric procedure must be pointed out: the
pressure measurement requires a concentration that is as high as
possible (2 mol/L; four-fold stoichiometric surplus! [18]) for rapid
absorption of the carbon dioxide, while the concentration of the
caustic soda solution of the titrimetric procedure requires a surplus
that is as low as possible (higher resolution!).
3.8 Methods for validation in routine operation:

test resource monitoring, control cards, intra-laboratory tests
The operating steps described above are used to determine the
principle utilizability of the measuring system and to obtain data on
this, with which system-dependent scattering margins of the measured
values can be calculated. If the procedure has been adopted in routine
operation, it must be ensured that the test system also provides "valid"
results in each test. For this purpose, control measures that supply this
proof with acceptable costs must be implemented and that each
laboratory can stipulate for itself individually. The following measures
can be used for this purpose:
1. Regular test resource monitoring, e.g. using the OxiTop PM [22]
calibration tablett or comparable methods, for example, at half-
yearly or yearly intervals with documentation of the results. If
unexpected problems occur, it may be useful to repeat the test at
short intervals of time. In this context, one should also take
advantage of the possibility to prove the operability of the time
measurement of the system.
12
2. Regular test resource monitoring of the pressure sensors by means

of injection technique. This procedure is less expensive and is
used to test the pressure sensors, not the whole system.
3. For the use of control cards for the workday monitoring of the
measuring system, as is usual for many water analytical
procedures within the scope of quality control, there is no
preparation that appears practical at the moment.
4. Intra-laboratory tests can provide a good tool for quality control.
However, the results achieved so far and the great variety of
evaluation options [23, 24] indicate that a great deal of
standardization work still needs to be carried out in this field.
In the lecture, the subjects covered by this text will be complemented by further diagrams.
4. Bibliography
[1] DIN 38409 Teil 51 (1987). Bestimmung des Biochemischen
Sauerstoffbedarfs in n Tagen nach dem Verdnnungsprinzip
(Verdnnungs-BSB).
In: Deutsche Einheitsverfahren zur Wasser-, Abwasser- und
Schlammuntersuchung (H 51). 18. Lieferung. VCH, Weinheim,
FRG und Beuth-Verlag, Berlin, FRG.
[2] DIN 38409 Teil 52 (1987). Bestimmung der Sauerstoffzehrung
in n Tagen. In: Deutsche Einheitsverfahren zur Wasser-,
Abwasser- und Schlammuntersuchung (H 52). 18. Lieferung.
VCH, Weinheim, FRG und Beuth-Verlag, Berlin, FRG.
[3] Isermeyer, H. (1952). Eine einfache Methode zur Bestimmung
der Bodenatmung und der Karbonate im Boden.
Z. Pflanzenern. Bodenk. 56: 25-38
[4] Alef, K. (1995). Soil respiration.
In: Alef, K., Nannipieri, P. (eds.). Methods in applied soil
microbiology and biochemistry. Academic Press, London, UK:
214-219.
[5] DECHEMA (1992). Labormethoden zur Beurteilung der
biologischen Bodensanierung.
2. Bericht des interdisziplinren Arbeitskreises
"Umweltbiotechnologie - Boden", Frankfurt, FRG
[6] DECHEMA (1995). Biologische Testmethoden fr Bden.
4. Bericht des interdisziplinren Arbeitskreises "Umweltbio-
technologie - Boden", Frankfurt, FRG
[7] Wagner, I., Fink, W. (1996). Ein quecksilberfreies BSB-
Mesystem zur Bestimmung des Biochemischen Sauerstoff-
bedarfs. Korresp Abw 43: 517-522.
13
[8] Sommer, I. (1996). Entwicklung und Prfung eines neuen

Mesystems zur Bestimmung der Bodenatmung mit
anschlieenden Versuchen zur Korrelation mit der Mikroorga-
nismenzahl. Diplomarbeit, Fachhochschule Gieen-Friedberg,
Fachbereich KMUB, Wiesenstrae 14, 35390 Gieen.
[9] Mner, R. (1997). Weiterfhrende Untersuchungen zur
Anwendbarkeit des OxiTop-OECD-Mesystems zur
Bestimmung der Bodenatmung. Diplomarbeit, Fachhochschule
Gieen-Friedberg, Fachbereich KMUB, Wiesenstrae 14,
35390 Gieen.
[10] Robertz, M., Eckl, S., Muckenheim, T., Webb, L. (1997).
Kostengnstige Methode zur Bestimmung der Bodenatmung
belasteter und unbelasteter Bden. Applikationsbericht
AL 97004, Forschungszentrum Jlich, FRG.
[11] Platen, H., Sommer, I. (1997). Bestimmung der
Atmungsaktivitt von Bden. TerraTech 3/1997: 23-25.
[12] Gesetz zum Schutz vor schdlichen Bodenvernderungen und
zur Sanierung von Altlasten (Bundesbodenschutzgesetz).
BGBl. Teil I, S. 502 ff vom 24.3.1998.
[13] DIN 19737 (Entwurf). Bodenbeschaffenheit - Laborverfahren
zur Bestimmung der mikrobiellen Bodenatmung.
Beuth-Verlag, Berlin, FRG. In Bearbeitung durch
Normenausschuss Wasserwesen I B 4/UA 1.
[14] Christen, H.R. (1977). Grundlagen der allgemeinen und
anorganischen Chemie. Sauerlnder, Aarau, CH. S. 173.
[15] Betriebsanleitung System OxiTop-Control.
WTW Weilheim, FRG BA31114/08.97/Pro/OxiTopControl-1
[16] DIN 38414 Teil 2 (1985). Bestimmung des Wassergehaltes
bzw. des Trockenrckstandes bzw. der Trockensubstanz.
In: Deutsche Einheitsverfahren zur Wasser-, Abwasser- und
Schlammuntersuchung. 15. Lieferung, Beuth-Verlag, Berlin,
FRG und VCH, Weinheim, FRG
[17] hlinger, R. (1993). Bestimmung der maximalen
Wasserkapazitt im Laborversuch. In: Schinner, F., hlinger,
R., Kandeler, E., Margesin, R. (Hrsg.). Bodenbiologische
Arbeitsmethoden, 2. Auflage, Springer, Berlin, FRG, S. 345.
[18] Platen, H., Wirtz, A. (1999). Applikationen zur Analytik Nr. 3:
Bestimmung geringer Atmungsaktivitten in Bden mit dem
Messsystem OxiTop-Control. 2. aktualisierte Auflage. FH
Gieen-Friedberg, FRG und WTW Weilheim, FRG.
Bestimmung der Atmungsaktivitt von Bden und Feststoffen
mit dem Messsystem OxiTop-Control. Standardprfansatz.
14
2. aktualisierte Auflage. FH Gieen-Friedberg, FRG und WTW

Weilheim, FRG.
Messung der Atmungsaktivitt von Bden mit dem Messsystem
OxiTop-Control. Grundlagen und Verfahrenskenngren.
2. aktualisierte Auflage. FH Gieen-Friedberg, FRG und WTW
Weilheim, FRG.
[21] Muckenheim, T., Groeneweg, J., Webb, L., Robertz, M. (ohne
Jahresangabe). Bestimmung der Bodenatmung
unterschiedlicher Bodenmaterialien mittels BOD/BSB-Sensor.
Informationsschrift des Forschungszentrums Jlich GmbH und
der Firma Aqualytic, Neu-Isenburg.
[22] WTW GmbH (ohne Jahresangabe). OxiTop PM
Kalibriertablette. Prfvorschrift fr die Prfung der
Funktionsfhigkeit des Messgerts. WTW, Weilheim, FRG.
[23] Institut WAR an der TU Darmstadt (1999): Arbeitskreis
Alternativparameter im BMBF-Verbundvorhaben "Mechanisch-
biologische Behandlung von zu deponierenden Abfllen".
Darmstadt, FRG.
[24] Fraunhofer-Institut fr Umweltchemie und kotoxikologie
(2000). kotoxikologische Testmethoden fr Bden -
Diskussion der Ringtestergebnisse. Schmallenberg, FRG.
[25] Platen, H., Bauer, S. (1996). Entwicklung der Bodenatmung in
einem Ackerboden nach Kontamination mit Diesel im
Laborversuch. In: In-situ-Sanierung von Bden, 11. Dechema-
Fachgesprch Umweltschutz, Frankfurt, FRG: 229-239.
15

Soil respiration measurements using the OxiTop Control
Soil respiration measurements with the

OxiTop Control experimental report
of a service provider
Sibylle Wachter, GAB Niefern
1. Introduction
Within the scope of the qualification procedure for plant protectives, a
check is required of the substances to be tested for effects on the soil
microflora. Many agents get onto or into the soil during their use,
whereby negative effects cannot be ruled out. Consequently, checks
must be carried out on the effect of the agent on the microbial activity,
above all with regard to the preservation of the soil fertility.
The effect of plant protectives can be analyzed using several
parameters. This generally concerns the following:
1. Metabolism activity of the microbial biomass
Short-term respiration:
The short-term respiration characterizes the ability of the soil
microflora to react to the addition of glucose within a few hours.
Dehydrogenase activity
2. Metabolically active biomass

Nitrogen balance
The effects of the substances to be tested for nitrification are
analyzed by the addition of lucerne.
Within the scope of the guideline for the official analysis of plant
protectives, the effect on the activity of the soil microflora (BBA,
1990), the determination of the short-term respiration parameters
was necessary.
2. Objective of the study

The short-term respiration parameter was first carried out using
Sapromat systems (model B 12, Voith company) known from BOD
determination. Contingent on the large-scale short-term respiration
studies to be performed in the GAB biotechnology, a search was made
for a simpler or better suited measuring system for this measuring
activity with which these studies could be carried out without
additional personnel and with acceptable investment. In addition to a
1st Symposium on Biological degradability.. 26th September, 2000 Theme 7: Sibylle Wachter

simpler management of a multitude of samples, the alternative

measuring system should be dependable, reliable and safe.
Furthermore, the system must meet the requirements of the GLP
(validation, data collection, archiving, etc.) and allow a parallel
measurement of a minimum of 12 measuring samples.
3. Method of proceeding
In pre-trials, the OxiTop Control measuring system was tested with
various vessel sizes and fill volumes. Furthermore, any feasible or
existing air-conditioning cabinets, air-conditioned rooms and water
baths for incubation at 20C were tried out.
As described below, extensive comparison measurements were carried
out between the Sapromat and OxiTop Control measuring systems.
The comparisons were made using two soil types. These can be
characterized as loamy sand and sandy loam. The measurements were
carried out using a soil treated with the herbicide Herbogil liquid in
direct comparison to a completely untreated soil. The herbicide is
commercially available as Dinoterb formulation and has a well-known
inhibitory effect on the soil microflora.
4. Material and method
4.1 Principle of the trial

The short-term respiration characterizes the ability of the soil
microflora to metabolize a defined quantity of glucose within a few
hours. The quantity of glucose added is chosen in the run-up in the
form of a glucose optimization.
The effect of the test substance on the short-term respiration is
measured on two different types of soil. The soil is incubated in the
dark at 20 2C for a period of 28 days. The short-term respiration of
the soil samples were determined after 3 hours, 14 days and 28 days
after application of the test substance or the solutions.
The oxygen consumption is measured immediately after the addition
of glucose for at least 20 hours at 20 2C.

4.2 Types of soil

For the realization of the trial, 2 different soil types were used.
According to the guideline, the soils must meet the following
conditions:
Soil 1
Loamy to clayey sand (amount of sand not more than 70%),
0.5 1% org. C and pH value of 5.5 7.0.
Soil 2
Loamy silt or loam,
1 3% org. C and pH value of 6.0 7.5.
The soil sample is to be taken from agricultural long-term productive
areas. No plant protective must have been spread on the areas selected
for the soil sample for at least a year beforehand. Furthermore, it must
be ensured that no organic fertilizer was used for at least a half year
beforehand and no mineral fertilizer was used for at least three months
beforehand. Apart from that, the mineral fertilizer is to be used
according to the requirements of the cultivated plants.
The soil was characterized before the start of the study. The results are
summarized in the following table.
Table 1: Soil characterization

Type 1 Type 2
(BBA 2.3)
Soil type Loamy sand Sandy loam
Dry weight (%) 89.3 77.6
pH 6.9 6.9
Organic C (%) 0.86 2.98
Humus (%) approx. 1.72 approx. 5.96
% microbial biomass (mg C/100 g TS), 10.4 47.6
calculated from the respiration activity
NH4+-N (mg/100 g TS) 0.29 0.28
NO3 --N (mg/100 g TS) 1.98 7.15
NO2--N (mg/100 g TS) 0.15 *
WHC ** (mL H2O/100 g TS) 33.4 56.0
Loam (%) < 2 m 9.1 6.2
Fine 2 - 6 m 3.4 7.4
SILT (%) Agent 6 - 20 m 7.4 24.6
Coarse 20 - 63 m 13.0 49.7
Sand (%) 63 - 2000 m 67.1 12.1
* = under the determination limit

4.3 Method
As already mentioned, soil microflora studies are called for by the
relevant qualification authorities within the scope of a qualification
procedure in plant protectives. For the realization of these studies, the
short-term respiration is determined according to the relevant BBA
guideline. The sample preparation for a study of this type is usually
made with each of 3 parallel samples, the control substance
(untreated), the reference substance (Dinoterb formulation) and the
substance under test. For the validation of the OxiTop Control
system, different pre-trials of only two groups (*without test
substance) with 3 parallel samples each (repeats) were carried out for
the determination of this parameter:
Untreated soil ( control )
Soil treated with reference substance
(Dinoterb formulation).
4.3.1 Preparation of the soil and application of the test agent

The soil sample of approx. 20 kg is transported and stored in
appropriate PE tubs. Before the start of the study, the water content
and water holding capacity (WHC) of the soil is determined. Then the
quantity of water that is required to adjust the soil to a water content
of 40 60 % of the maximum WHC according to instructions or
according to the relevant guideline is calculated. Also the quantity of
glucose with which an optimum short-term respiration rate can be
achieved is determined before the start of the study.
Preparation of the reference substance stock solution.
(The concentration of the stock solution corresponds to a 5-fold
consumption quantity of the agent (20 L/ha). The quantity of the agent
to be applied is calculated based on a penetration depth of 5 cm and a
soil density of 1.5 according to the instructions of the guideline).
Preparation of the test substance* stock solution according to the
guideline
Partial quantities of 1 2 dm3 soil are each separated from the
dispensing cask of the soil and adjusted to the necessary WHC
according to study 3.
1. Partial quantity remains untreated (control substance)
2. Partial quantity with the reference stock solution applied
(reference substance)
3. Partial quantity* with the test stock solution* (test substance)
applied.
The added solutions must be thoroughly mixed with the soil.
After the application of the solutions to the respective partial quantity

of soil (time frame up to 3 hours) each of 3 parallel samples (repeats)

are immediately poured into the graduated flasks. Immediately before

the start of the measurement the corresponding quantity of glucose is
added to the measuring samples and well mixed.
The remaining partial quantities are now transferred to glass stock
bottles of 1000 2000 ml. The bottles are weighed to determine their
initial weight and the lids of the bottles are placed loosely on the
bottles. The filled glass bottles are incubated in the dark at 20 2 C.
After approx. every 7 days, the samples are re-moistened.
A sample is taken from these glass bottles after:
14 days and
28 days.
On each of these dates the following parameters of the samples are

determined:
Dry weight
pH value
Short-term respiration
4.4 Measuring systems
4.4.1 Sapromat
For the measurement of the samples, 100 g soil were poured into the
vessels, the relevant quantity of glucose (500 mg /100g soil) added,
mixed with the soil and incubated at 20C for at least 20 hours. Soda
lime was used as the CO2 absorber. The oxygen consumption graph
was plotted by means of a recorder. The graph of the oxygen
consumption (mg O2/h) showed an increase within the first 12 hours.
The principle of the Sapromat B12 (Voith, Heidenheim) is that the
oxygen consumed by the microorganisms is directly supplied via an
electrolytic cell. The system consists of 12 measuring units that are
connected with one another by tubes, a water bath with combined
heating and cooling system and an analog recorder. Each measuring
unit consists of a reaction vessel, an oxygen generator (electrolytic
cell) and a differential pressure switch. The microorganisms in the
reaction vessel require oxygen for the organic degradation and emit
CO2 in the headspace. The CO2 created by the microorganisms in
biogenic degradation processes is fixed by means of the CO2 absorber
and, thus, creates a partial vacuum in the closed system. The
differential pressure sensor records this partial vacuum and controls
the oxygen supply until the pressure conditions in the measuring unit
are equalized again. The oxygen that is created can be measured and
added up via the electrical energy required to equalize the pressure.
4.4.2 OxiTop Control

For the measurement of the samples, 200 g soil are poured into the
graduated flasks, the appropriate quantity of glucose (500 mg / 100 g
soil) is added, mixed and incubated for 24 hours at 20 C. Soda lime
was also used as CO2 absorber.

In the OxiTop Control system, the microorganisms draw the oxygen

required for the biogenic degradation from the air supply found above
the sample. The measuring units (graduated flasks with measuring
heads) were incubated at 20 C using the same water bath with a
heating and cooling system as described in section 4.4.1. The system
consists of the graduated flasks (500 ml Duran 50 glass) with OxiTop
adapters and CO2 absorber container, the OxiTop-C measuring heads
and the OxiTop Controller OC110. The CO2 created by the
microorganisms in biogenic degradation processes is absorbed by the
CO2 absorber in the headspace of the closed graduated flask. The
pressure difference resulting from this is stored by the measuring
heads in the specified measuring period of 24 hours as 360 measuring
points. The measuring head data that are read via the controller form
the basis of the calculation of the soil respiration (section 4.5)
4.5 Evaluation
The evaluation was carried out in both measuring systems within the
first 12 hours in the linear range of the measurement, i.e. usually
between 6 and 12 hours. The CO2 production is calculated from the
oxygen consumption; based on the stoichiometry of the O2
consumption and the CO2 production (1 mg O2 consumption
corresponds to 1.375 mg CO2). The values are calculated as the mean
value of 3 parallel samples (repeats). The inhibition of the agent is
calculated as the difference of the comparison of the untreated soil
group (control) and the treated soil group.
The evaluation of the Sapromat graphs was performed graphically
from the oxygen consumption or directly from the increase of the
oxygen consumption curves. As a result, this was very complicated,
time consuming and inaccurate. Adaptation of the existing instruments
to evaluation by computer was not possible in this model.
The calculation of the soil respiration via the OxiTop Control system
is carried out using the following soil respiration equation using the
differential pressure values that were determined.
The display and evaluation of the OxiTop Control system offers very
flexible possibilities. The measured pressure data can be read from the
measuring heads by infrared transmission at any time during the trial
with the controller. The pressure curve is immediately displayed on
the controller display and provides a rapid overview of the trial curve.
In addition, the Achat OC PC communication program provides
comfortable direct data transmission to the PC. Further data
processing is performed via the Quattro-Pro spreadsheet program.

Calculation of the soil respiration:
MR (O 2 ) Vfr
BA = p
R T mBt
BA = soil respiration [in mg O2 / kg DS]
MR (O2) = molar mass oxygen : 32000 mg/mol
Vfr = free gas volume [in L] (see equation 7 )
R= general gas constant: 83.14 L mbar mol-1 K-1
T= measuring temperature [in K]
mBt = mass of dry soil substance in the measuring preparation
p = pressure decrease in the measuring preparation [in mbar]
(Result specification under specification of the measuring period
and the incubation temperature
Example: Soil respiration (20C) = 360 mg O2/kg DS in 4 days)
Calculation of the free gas volume:
Vfr = Vges VAG VAM VBf
Vfr = free gas volume [in L]

Vges = total volume of the headspace in the measuring vessel [in L]
enclosed by the lid
(without soil, without absorption vessel, without
absorbing agent)
VAG = intrinsic volume of the vessel for the absorbing agent [in L]
VAM = intrinsic volume of the absorbing agent [in L]
VBf = volume of the moist soil [in L]
Calculation of the dry soil substance used:
mBt = mass of dry soil substance [in kg]

mBf = mass of moist soil substance [in kg]
DS = dry substance content [in %]
100%= correction term

4.6 Results
4.6.1 Pre-trials with different volumes of the reaction

vessels and different weights of soil
Table 2: Pre-trials with different vessel volumes and weights of soil

hPa/h % DS mg O2 (x 1.33)/ Mean Standard
Sample /100 h x 100 g DS value deviation
1.21 0.882 0.33
Control 1 1.22 0.880 0.33 0.33 0.01
(100 g soil, 250 ml vessel) 1.23 0.882 0.34
2.50 0.884 0.21
Control 2 2.01 0.878 0.17 0.24 0.05
(200 g soil, 250 ml vessel) 2.96 0.867 0.26
0.33 0.883 0.18
Control 3 0.34 0.881 0.19 0.19 0.01
(100 g soil, 500 ml vessel) 0.34 0.876 0.19
0.90 0.883 0.25
Control 4 0.85 0.881 0.23 0.24 0.01
(200 g soil, 500 ml vessel) 0.85 0.876 0.23
Formula: Increase x 1.33 x (mL test vessel - g soil /density soil - adsorber volume) /
(DS/100)/(g soil /100g soil )
4.6.2 Glucose optimization
Table 3: Maximum rate of the short-term respiration

Sample hPa/h % soil DS mg O2
g Glc / 100 g soil /100 h x 100 g DS
0 Glc 0.18 0.913 0.05
0.1 Glc 1.61 0.913 0.43
0.2 Glc 1.78 0.913 0.47
0.3 Glc 2.09 0.913 0.56
0.4 Glc 1.97 0.913 0.52
0.5 Glc 2.01 0.913 0.53
0.6 Glc 2.06 0.913 0.55

The graph of the glucose optimization is shown in figure 1.
Figure 1:
Oxygen consumption curves at different
concentrations of glucose
4.6.3 Results of the short-term respiration using

OxiTop Control
Table 4: Results of the short-term respiration using the OxiTop

Control system
mg CO2/h/100 g DS
Loamy sand Sandy loam
Time Control Herbogil Control Herbogil
liquid liquid
3h 0.37 0.44 1.70 1.91
14 d 0.27 0.22 2.01 1.95
28 d 0.27 0.17 2.14 1.77

The following diagrams 2 and 3 show the short-term respiration.
Figure 2:
Effects of Herbogil liquid on
the short-term respiration, measured by the
OxiTop Control (loamy sandy soil)
Figure 3:
the short-term respiration, measured using
the OxiTop Control (sandy loamy soil)
10

4.6.4 Results of the short-term respiration using the Sapromat
Table 5: Results of the short-term respiration using the Sapromat

mg CO2/h/100 g TS
Loamy sand Sandy loam
Time Control Herbogil Control Herbogil
liquid liquid
3h 0.56 0.48 2.67 2.49
14 d 0.48 0.44 2.44 2.04
28 d 0.54 0.33 2.33 1.74
Displays of the short-term respiration rates measured with the

Sapromat are given in figs. 4 and 5.
Figure 4:
the short-term respiration, measured with
Sapromat (loamy sandy soil)
Figure 5:
the short-term respiration, measured with the
Sapromat (sandy loamy soil)
11

5. Summary and conclusion

The OxiTop Control system was tested in order to establish a new
measuring system for the determination of the short-term respiration
in direct comparison with control substance and reference substance to
the Sapromat (B12). The results confirm the reproducibility of the
results. In contrast to the Sapromat, the OxiTop Control system
provides a variable working method and can be extended as a result of
its modular structure. Our experience indicates that the reliability of
the measuring system, the measurement security and measurement
accuracy is better than in the Sapromat. In addition, this system
requires no or only marginal running maintenance costs. We
regard the fundamentally lower system and investment costs and the
advantage of test resource monitoring supported by the system with
independent verification according to GLP as a further advantage.
In our experience, the OxiTop Control system is exceptionally well
suited for the determination of the short-term respiration in soils
because of its user-friendliness and safe handling.
The analyses carried out led to the result or to the decision by the
GAB to switch over the complete short-term respiration measuring
operation from the Sapromat to the OxiTop Control system due to its
economical price/performance ratio, its fundamentally simpler, more
user-friendly and faster working method where it has been operated
successfully.
6. Bibliography
AUSWIRKUNGEN AUF DIE AKTIVITT DER BODENMIKROFLORA:
Biologische Bundesanstalt fr Land- und Forstwirtschaft Bundesrepu-
blik Deutschland, Richtlinien fr die amtliche Prfung von
Pflanzenschutzmitteln, Teil VI, 1-1 (2.Auflage), Mrz 1990, issued by
the Abteilung fr Pflanzenschutzmittel und Anwendungstechnik der
Biologischen Bundesanstalt Braunschweig
DEUTSCHE EINHEITSVERFAHREN ZUR UNTERSUCHUNG VON

SCHLMMEN, BDEN, SEDIMENTEN UND ABFALL:
Bestimmung des Wassergehaltes und des Trockenrckstandes, bzw.
der Trockensubstanz, DIN 38414
MALKOMES, H.-P.: Einflsse von Pflanzenschutzmitteln auf Boden-

mikroorganismen und ihre Leistungen. Berichte ber Landwirtschaft,
Sonderheft 198, 134 - 147 (1985)
12

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Biological Degradability:

The significance of standardized

Dr. Udo Pagga, BASF AG Ludwigshafen

1. Biological degradation in the environment

The most important biological degradation processes in the natural

Biological degradation processes take place not only in fresh water,

The forms of colonization and activities of man have, over a long

Industrialization has also increasingly resulted in the release of

2. Test of biological degradability

Test period (d)

In addition to the degradation of organic substances, there are also

3. Standardized degradation test methods

2) Static (batch test), semi-continuous and continuous;

In a typical aerobic, aquatic batch test of easy biological degradability,

or an additional supply of oxygen. As the test method for wastewater,

4. Evaluation of the results and consequences of degradation

Table 1: Evaluation of the test results

DOC tests BOD/CO2 tests Evaluation

>70 % >60 % biologically degradable and, acc. to OECD criteria,

20-70 % 20-60 % moderately or partly biologically degradable under the test

>20 % >20 % indications of potential degradability acc. to OECD criteria

<20 % <60 % poorly biologically degradable under the test conditions

If, in the use of the DOC as an analysis parameter, no unequivocal

also result that classifies a substance as readily degradable although a

A further example for the consequences that result from the

5. Quality management and supply source of method regulations

Overview of standardized methods for biological degradation

Degradation test OECD EG ISO DIN EN ISO

Carrying out the manometric

Dr. Peter Reuschenbach, BASF AG Ludwigshafen

1. The search for a new type of respirometer

Because, however, the Sapromats are relatively old in their basic

2. The manometric respiration test

The measurement of biological degradability in two types of

apparatus has no system for the subsequent supply of oxygen. Hence,

3. The biochemical oxygen demand in blank value preparations

3.2 The graph of the biochemical oxygen demand in blank value

4. The search for a reference substance for the

4.1 The reference substance aniline

4.2 Differences in the degradation of aniline in the most

approximately a week earlier in this test than, e.g. in the manometric

4.3 Compliance with validation criteria in the degradation of the

4.4 The degradation of the reference substance aniline in the

4.5 Nitrification in the degradation of the reference substance

4.6 Nitrification in the degradation of the reference substance

system, but occurred in practically all of the other analyzed standard

Tab. 2: Total nitrogen concentration of nitrite and nitrate nitrogen

4.7 The degradation of aniline in the presence of the nitrification

4.8 The degradation of the reference substance benzoic acid in

4.9 Prospects the multi-component measuring system

As in the OxiTop Control test system, the carbon dioxide in the

5. The manometric respiration test in the presence

6. Biological degradability of substances in the comparison of the

prevailing high-energy entry in the two systems or were caused by

Tab. 3: Evaluation of the biological degradability of substances in

Substance Evaluation of the biological degradability

The reference substances, aniline and benzoic acid, were

The nitrification of aniline could be suppressed by adding the

Further development of the biological

Prof. Dr. Uwe Strotmann, Fachhochschule Gelsenkirchen

1. Principles of aerobic biological degradation

2. The OECD 301 test systems

Decrease in dissolved organic carbon (DOC): 301 A, E

On closer inspection of the parameters, it becomes apparent that the