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Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June-August 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Substance type: organic
- Physical state: off-white crystalline powder

Study design

Oxygen conditions:
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge : municipal sewage treatment plant
- Concentration of suspended solids was 3.5 g/l in the concentrated sludge
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 31.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test duration 28 days (last CO2-measurement on the 29th day).
During the test period the test media were aerated and stirred continuously.

Test vessels 2 litre all-glass brown coloured bottles.

Milli-RO / Milli-Q water Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

Preparation of bottles:
Pre-incubation medium The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).

Preparation At the start of the test (day 0) test and reference substance were added to the bottles containing the microbial organisms and mineral components.
The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Determination of CO2
Experimental CO2 production The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul), Merck, Darmstadt, Germany).

Measurements Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the 28th day, the pH of all test suspensions was measured and 1 ml of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

Theoretical CO2 production The theoretical CO2 production was calculated from the molecular formula.

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Key result
% degradation (CO2 evolution)
Sampling time:
10 d
Key result
% degradation (CO2 evolution)
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
71% biodegadability within 14 Days

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
b-Nicotinamide-adenine Dinucleotide II (nadide) was readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992.

Nadide was tested in duplicate at 31.5 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. The Theoretical CO2production (ThCO2) of nadide was calculated to be 1.39 mg CO2/mg.


The study consisted of six bottles:

-         2 inoculum blanks (no test substance),

-         2 test bottles (nadide),

-         1 positive control (sodium acetate) and

-         1 toxicity control (b-Nicotinamide-adenine Dinucleotide II (nadide) plus sodium acetate).


nadide was easily soluble in water the test media were prepared using a stock solution of 1 g/l in Milli-RO water.The final stock solution was all clear and colourless. Aliquots of 63 ml of the stock solution were added to the test substance bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms (final volume: 2 litres). The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on the 29thday).


The relative biodegradation values calculated from the measurements performed during the test period revealed 81 and 79% biodegradation nadide, for A and B, respectively. Furthermore, nadide of at least 60% was reached within a 10-day window. Thus, the criterion for ready biodegradability was met. In the toxicity control, nadide was found not to inhibit microbial activity.


In conclusion, nadide was designated as readily biodegradable.