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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:
from 2017-10-26 to 2018-01-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
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: Activated sludge, microorganisms from a domestic waste water treatment plant. Supplied on 27 October 2017 (seven days before the main test).
- Storage conditions: aerating (2 L/minute) of the activated sludge in mineral medium at 20.1 - 21.7 °C
- Storage length: 7 days
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. 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 5 g dry material per litre was mixed with test water (mineral medium) and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.53, just before use: 7.31.
- Pretreatment: The inoculum was not pre-adapted to the test chemical.
- Concentration of sludge: 5 g dry material per litre
- Initial cell/biomass concentration: ~1E8/L
- Filtered: yes
- Type and size of filter used: cotton wool
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
test mat.
Initial conc.:
7.11 mg/L
Based on:
COD
Remarks:
mg O2/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium used. Prepared according to OECD 301.
- Test temperature: 20.0 - 20.1 °C
- pH: 7.43
- pH adjusted: no
- Aeration of dilution water: yes, for 20 minutes
- Suspended solids concentration: 5 g dry material/L
- Continuous darkness: yes
- Oxygen concentration of the test water: About 8 - 9 mg/L (was measured at the start of the test and found to be 8.30 mg/L).

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
- Number of culture flasks/concentration:
16 (+ 2 reserve) bottles containing the test item and inoculum;
16 (+ 2 reserve) bottles containing the reference item and inoculum (procedure control);
- Measuring equipment: The oxygen concentration was measured with an O2 electrode.

SAMPLING
- Sampling frequency: in all duplicate bottles in all groups on days 0, 2, 5, 7, 12, 14, 21 and 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 16 (+ 2 reserve) bottles containing only inoculum (inoculum control)
- Toxicity control: 16 (+ 2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)
Reference substance:
benzoic acid, sodium salt
Remarks:
3 mg/L (test. mat.), 5.01 mg O2/L (ThOD)
Preliminary study:
No toxic effect of the test item was found at the investigated concentration - 3.0 mg/L.
Key result
Parameter:
% degradation (O2 consumption)
Value:
12.2
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of the test item reached a mean of 12.2 % after 28 days based on its COD.
The pass level for ready biodegradability is the removal of 60 % ThOD (in the present case COD) in a 10-day window. Minimal biodegradation of the test item occurred in this study; the value remained far below the pass level; therefore, the test item was considered to be not ready biodegradable.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to a mean of 79.7 % after 14 days, and to a mean of 82.8 % after 28 days of incubation, based on its ThOD. The biodegradability reached its plateau on about the 12th day.

Correction for Oxygen Uptake for Interference with Nitrification:

Because of the N-containing test item, the oxidised nitrogen (nitrate and nitrite) concentrations were determined following each oxygen measurement with photometric method using nitrite and nitrate cell tests. The LOQ (Limit Of Quantification) of the measurements was 0.03 mg NO2/L and 0.4 mg NO3/L, respectively.

The measured quantities of nitrite in the inoculum control, test item and toxicity control samples were below the LOQ in the 0-day, 7-day, 14-day and 21-day samples. The nitrite concentration of the 28-day samples in the case of inoculum control was in average 0.11 mg/L, in the case of test item was in average 0.10 mg/L and in the case of toxicity - control in average 0.18 mg/L.

The nitrate concentration was less than 0.4 mg/L in the 0-day, 7-day, 14-day, 21-day and 28-day in the inoculum control, test item and toxicity control samples.

According to the referred OECD 301 guideline (Annex V) the oxygen consumed in nitrate formation approximates 4.57x increase of nitrate-N concentration, and the oxygen consumed in nitrite formation is 3.43x increase of nitrite-N concentration.

In this study the change of the measured dissolved oxygen concentrations in the inoculum control, toxicity control and test item bottles did not correspond to the calculated consumed oxygen of ammonium oxidation processes. The oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population. Therefore, any correction of the measured dissolved oxygen concentrations was considered as not necessary.

Biodegradation of Toxicity Control

In the toxicity control containing both, the test item and the reference item, a mean of 35.5 % biodegradation was noted within 14 days and a mean of 37.1 % biodegradation was determined after 28 days of incubation.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The percentage biodegradation of the test item reached a mean of 12.2 % after 28 days based on its chemical oxygen demand. Therefore, the test item is considered to be not readily biodegradable.
Executive summary:

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Closed bottle test according to OECD 301D, Regulation (EC) No. 440/2008 Method C.4 -E and EPA OPPTS 835.3110 and under GLP. The test item was tested at a concentration of 3 mg/L in triplicates, corresponding to a COD of 7.11 mg O2/L. The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. As the substance contains nitrogen, the effect of nitrification on the oxygen uptake was also measured. It was determined that the nitrification doesn't play a role on the oxygen uptake of the the test item, therefore, no correction due to nitrification was performed. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The biodegradation of the test item came to 12.2 % after 28 days. In conclusion, the test item is not readily biodegradable as the pass level of 60 % degradation was not reached within the 28 days period of the study.

Description of key information

The percentage biodegradation of the test item reached a mean of 12.2 % after 28 days based on its chemical oxygen demand. Therefore, the test item is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Closed bottle test according to OECD 301D, Regulation (EC) No. 440/2008 Method C.4 -E and EPA OPPTS 835.3110 and under GLP. The test item was tested at a concentration of 3 mg/L in triplicates, corresponding to a COD of 7.11 mg O2/L. The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. As the substance contains nitrogen, the effect of nitrification on the oxygen uptake was also measured. It was determined that the nitrification doesn't play a role on the oxygen uptake of the the test item, therefore, no correction due to nitrification was performed. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The biodegradation of the test item came to 12.2 % after 28 days. In conclusion, the test item is not readily biodegradable as the pass level of 60 % degradation was not reached within the 28 days period of the study.