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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-12-02 to 2014-12-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage plant Rossdorf, Germany.
- Conditioning: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water (see 6.5) and aerated overnight. This suspension was used for the experiment.
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
103.1 mg/L
Based on:
test mat.
Initial conc.:
172.4 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: According to test guidelne
- Test temperature: 22 °C± 1 °C
- pH: 7.6 (measured at the start of the test); 7.5 to 7.9 (measured at the end of the test)
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BSB/BOD-Sensor-System (Aqualytic Dortmund, Germany); Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Preparation of Test Flasks: The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used.
- Number of culture flasks/concentration:
2 replicates for the test substance
2 replicate for the inoculum control
1 replicate for the procedure control with reference substance
1 replicate for thr abiotic control
1 replicate for the toxicity control
- Measuring equipment:
Measurement of Oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany) each day.
Temperature: The temperature was recorded by means of the automated software AMR Wincontrol®.
pH value: 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.
- Details of trap for CO2: The closed test flasks were incubated in a climatised room under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: Yes
- Procedure control: Yes
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
73
Sampling time:
28 d
Details on results:
The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days. The 10-day window started at day 2, when the biodegradation was 20%. At the end of the 10-days window at day 12, the biodegradation was 58% and therefore the 10 day window criterion was not passed. The degradation of the test item increased till day 25 when a plateau was reached, the maximum degradation was 76%; at the end of the test at day 28 the degradation was 73% (ThODNH4).
Therefore, the test item is considered to be readily biodegradable, but failing 10-day window.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 81% after 14 days and to 82% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Validity Criteria of the Study

Inoculum Control:

The oxygen demand of the inoculum control (medium and inoculum) was 30 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.

pH-Value:

The pH-value of the test item flasks at the end of the test was 7.5 and 7.6 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

Reference Item:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60% after 3 days of incubation.

Test Item:

The difference of duplicate values for the degradation of the test item at the end of the 10-day window, at the plateau and at the end of the test at day 28 differed by less than 4%. The validity criterion was fulfilled.

Toxicity Control:

If in a toxicity test, containing both the test substance and a reference compound less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was 72% at day 14; the test item was not inhibitory.

Biodegradation in the Toxicity Control

In the toxicity control containing both, the test item and the reference item sodium benzoate, 72% (ThODNH4> biodegradation was noted within 14 days and 71% after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Abiotic Control

The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no use to correct the degradation of the test item and toxicity control.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The degradation rate of the test item did not reach 60% within the 10-day window, but after 28 days. Therefore, the test item is considered to be readily biodegradable, but failing 10-day window.
Executive summary:

The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days according to OECD guideline 301F. The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 72% (ThODNH4) biodegradation was noted within 14 days and 71% biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days. The 10-day window started at day 2, when the biodegradation was 20%. At the end of the 10-days window at day 12, the biodegradation was 58% and therefore the 10 day window criterion was not passed. The degradation of the test item increased till day 25 when a plateau was reached, the maximum degradation was 76% at days 25 to days 27; at the end of the test at day 28 the degradation was 73% (ThODNH4).

In conclusion, the degradation rate of the test item did not reach 60% within the 10-day window, but after 28 days. Therefore, the test item is considered to be readily biodegradable, but failing 10-day window.

Description of key information

In a manometric respirometry test according to OECD guideline 301F, the degradation rate of the test item did not reach 60% within the 10-day window, but after 28 days. Therefore, the test item is considered to be readily biodegradable, but failing 10-day window (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window
Type of water:
freshwater

Additional information

The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days according to OECD guideline 301F. The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 72% (ThODNH4) biodegradation was noted within 14 days and 71% biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days. The 10-day window started at day 2, when the biodegradation was 20%. At the end of the 10-days window at day 12, the biodegradation was 58% and therefore the 10 day window criterion was not passed. The degradation of the test item increased till day 25 when a plateau was reached, the maximum degradation was 76% at days 25 to days 27; at the end of the test at day 28 the degradation was 73% (ThODNH4).

In conclusion, the degradation rate of the test item did not reach 60% within the 10-day window, but after 28 days. Therefore, the test item is considered to be readily biodegradable, but failing 10-day window (reference 5.2.1-1).