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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Jan - 13 Feb 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
according to
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
GLP compliance:
yes (incl. certificate)
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from the aeration tank of domestic waste water treatment plant, Rossdorf, Germany
- Storage conditions: aerobic
- Storage length: overnight
- Preparation of inoculum for exposure: The activated sludge used for this study was settled for 30 min before it was washed by centrifugation and the supernatant liquid phase decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was performed three times. The sediment of the last washing was resuspended in test water and aerated overnight until use. An aliquot of this final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per L (± 10%) were mixed with test water and then aerated until use. This suspension was used for the experiment.
- Concentration of sludge: 3.5 g/L on dry matter base (final sludge concentration in test flasks: 28.7 mg sludge/L)

Duration of test (contact time):
34 d
Initial conc.:
204.6 mg/L
Based on:
other: ThODNH4
Initial conc.:
223.8 mg/L
Based on:
other: ThODNO3
Initial conc.:
101.8 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: Reconstituted Test Water, prepared from analytical grade salts.
- Test temperature: 22 ± 1 °C
- pH: 7.6 (at the start of the test), 7.5 - 7.7 (at the end of the test)
- Suspended solids concentration: 28.7 mg sludge/L
- Continuous darkness: Yes.

- Culturing apparatus: BSB/BOD-Sensor-System, Aqualytic, Dortmund, Germany
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD-Sensor-System, Aqualytic, Dortmund, Germany
- Test performed in open system: The test flasks were closed gas-tight by a measuring head.
- Details of trap for CO2 and volatile organics if used: A potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide.

- Sampling frequency: The change of pressure in the test flasks was measured by means of a manometric method each day. The temperature was recorded by means of the automated software AMR Wincontrol. pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

- Inoculum blank (activated sludge in test water): 2 replicates:
- Abiotic sterile control: 1 replicate: test item and CuSO4 in test water.
- Toxicity control: 1 replicate: test item, reference item and activated sludge in test water.
- Procedure control: 1 replicate: reference item and activated sludge in test water.
Reference substance:
benzoic acid, sodium salt
% degradation (O2 consumption)
Sampling time:
34 d
Remarks on result:
other: ThODNO3
% degradation (O2 consumption)
Sampling time:
34 d
Remarks on result:
other: ThODNH4
Details on results:
READY BIODEGRADABILITY OF THE TEST ITEM: The degradation rate of the test item did not reach 60% within the 10 -day window or after 28 or 34 d. Therefore, the test item is not considered readily biodegradable.
BIODEGRADATION IN THE TOXICITY CONTROL: In the toxicity control containing both the test item and the reference item (sodium benzoate), 37% (ThODNH4) biodegradation was recorded within 14 d and 41% (ThODNH4) biodegradation after 28 d of incubation (35% and 39% based on ThODNO3). According to the test guidelines, the test item is not considered inhibitory to aerobic activated sludge microorganisms because degradation was > 25% within 14 d.
ABIOTIC CONTROL: The oxygen demand in the abiotic control was 0 mg/L throughout the test duration.
Results with reference substance:
The reference item (sodium benzoate) was sufficiently degraded to 87% after 14 d and to 96% after 28 d of incubation. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Table 1. Validity criteria of OECD

Criterion from the guideline

Study results

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

The difference of duplicate values at day 34 differed by 8% based on ThODNH4 (6% based on ThODNO3).


Percentage degradation of the reference compound has reached the pass levels by day 14.

The reference item sodium benzoate was degraded to more than 60% after 5 days of incubation.


The oxygen uptake of the inoculum blank is normally 20-30 mg O2/L and should not be greater than 60 mg/L in 28 days.

The oxygen demand of the inoculum control was 23 mg O2/L


Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable

Description of key information

Not readily biodegradable (9% after 34 d,based on ThODNO3 and 10% based on ThODNH4 OECD 301 F)

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable

Additional information

There is one GLP study available, which investigated the ready biodegradability of the substance according to OECD guideline 301 F.

In a manometric respirometry test, a nominal concentration of 101.8 mg/L test item was inoculated with activated sludge of a municipal sewage treatment plant. Degradation was followed by continuous measurement of the O2 consumption of the inoculum. The amount of O2 taken up by microbial population during biodegradation of the test item (corrected for the value in the blank control) was expressed as percentage of the theoretical O2 demand, ThOD, and serves as the measure for biodegradation. The test item contains nitrogen. Therefore, the evaluation of biodegradation has to be based on ThODNH4 and ThODNO3.

ThOD NH4 is the total amount of oxygen required to completely oxidise a substance. ThODNO3 is the total amount of oxygen required to completely oxidise a substance, assuming nitrification occurs. Based on ThODNH4 a mean biodegradation of 10% was reached at day 22. Whereas based on ThODNO3 a mean biodegradation 10% was reached at day 24. The test period was extended to 34 days. The mean biodegradation at test end after 34 days was 10% (ThODNH4) and 9% (ThODNO3). The procedure control reached 87% biodegradation after 14 d, thus confirming the suitability of the inoculum and test conditions. The toxicity control showed no significant toxic effects on the microbial population since more than 25% degradation occurred within 14 d. The abiotic sterile control confirmed that the test substance was not abiotically degraded by processes using O2 during the test (lack of oxygen consumption). Therefore, the substance is considered to be not readily biodegradable based on ThODNH4 and ThODNO3.