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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:
20 May 2016 - 18 June 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
certificate issued on 5 Nov 2015

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
C13 H9 N O5
Test material form:
Details on test material:
- Appearance: white solid
- Storage conditions: in refrigerator (2-8°C) protected from light
Specific details on test material used for the study:
Stability: stable under storage conditions
Carbon content: 60.2%

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Non-adapted activated sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant; sampling on 19 May 2016 (one day before start of the test)
- Storage conditions and length: the sludge was pre-conditioned for 1 day (aerated but not fed) to reduce the amount of
O2 consumed by the blank controls
- Preparation of inoculum for exposure: prior to the test the sludge was washed twice with tap water and once with mineral medium. After centrifugation, the sludge was suspended in test medium at a concentration twice of the final concentration.
- Initial cell/biomass concentration: 30 mg/L dry matter in the final mixture
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
100 other: mg O2/L
Based on:
Initial conc.:
58 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: Aerobic mineral salts medium prepared with ultrapure water, according to OECD 301F
- Additional substrate: no
- Test temperature: 22 ± 2°C, controlled at ± 1°C, in a thermostat cabinet
- pH: start: 7.2; end: 7.5 - 7.7 (blank controls: 7.3, toxicity controls: 7.2)
- pH adjusted: yes (in the beginning to 7.2 +/- 0.2) in blank control and procedure control
- Aeration of dilution water: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: not indicated

- Culturing apparatus: 510 ml glass bottles (tightly closed with manometric BOD measuring devices) containing a total volume of test solution of 200 ml. The bottles are equipped with stirring rods and butyl rubber quivers which contain 2 pellets of sodium hydroxide each to absorb the produced CO2 from the head space.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration 1 day prior to the test
- Measuring equipment: respirometric activity was measured as pressure change. Because the formed CO2 is adsorbed, O2 consumption leads to a pressure decrease, which is recorded by a manometric BOD measuring device (OxiTop®-C head; WTW Wissenschaftlich-Technische Werkstätten GmbH & Co. KG, DE-82362 Weilheim); DOC was determined with a Shimadzu TOC-Analyzer TOC-L CSH (Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach) using the non-purgeable organic carbon (NPOC) mode.
- Test performed in closed vessels: yes

- Sampling frequency: continuously
- Sampling method: manometric BOD measuring device (automatic)
- Sample storage before analysis: not applicable
- Other: at the end of the test, the pH was measured in all flasks except the abiotic sterile control

- Inoculum blank: yes, 2 replicates
- Abiotic sterile control: yes, 1 replicate
- Toxicity control: yes, 1 replicate
- Other: procedure control containing inoculum, test medium and sodium benzoate as ready biodegradable reference item (2 replicates)

STATISTICAL METHODS: no statistical analysis was performed
Reference substance
Reference substance:
benzoic acid, sodium salt
Purity: 99.8%; Carbon content: 58.3%; ThOD: 1.665 mg O2/mg

Results and discussion

% Degradationopen allclose all
Key result
% degradation (O2 consumption)
Sampling time:
28 d
% degradation (DOC removal)
Sampling time:
28 d
Details on results:
The biodegradability of the test item based on O2 consumption was calculated to be 85% after 28 days as compared to the theoretical oxygen demand (ThOD).The biodegradation reached 61% at the end of the 10-d window (i.e. within 10 days after
attainment of 10% degradation).
Biodegradation of the test item was observed after a lag-phase of about 5 days. The respective concentrations of organic carbon at the beginning (theoretical value based on the test item’s carbon content and the test concentration, as mg TOC/L) and at the end of the test (mean measured value of the two replicates, as mg DOC/L) were, respectively:
34.9 and 1.11 for the test units
<0.5 and 1.27 for the blank control
35.0 and 1.04 for the procedure control
The total elimination calculated based on dissolved organic carbon (DOC) measurements reached 100% for Phthaloyl-L-glutamic acid anhydride and 101% for sodium benzoate, respectively. This data confirm the biodegradability of the test item since the total elimination based on DOC was slightly higher as compared to the biodegradation based on BOD.

The procedure control sodium benzoate reached 86% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

At the applied initial test concentration of 58.0 mg/L the test item was not judged to have any inhibitory effect on the microbial population, since the biodegradation of the mixture (test item + reference item sodium benzoate) exceeded 25% within 14 days.

The test item was not abiotically degraded (by processes using O2) during the whole test period of 28 days in the absence of microorganisms as confirmed by the lack of oxygen consumption in the abiotic sterile control.

BOD5 / COD results

Results with reference substance:
86% biodegradation after 14 days

Any other information on results incl. tables

The O2 uptake values and degradation data are summarized in the tables attached under the ''Attached background material'' section.

The following validity criteria were fulfilled and therefore the test was considered valid:

1. The difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the 10-d window, or at the end of the test, as appropriate, must be less than 20% - actual: 4% at the end of the 10 -d window (day 20) and 0% at the end of the test (day 28)

2. The percentage of degradation of the reference item must reach the pass level of 60% by day 14 - actual: 86% by day 14

3. In the toxicity control, the degradation of the mixture test item + reference item must reach the pass level of 25% by day 14; otherwise, the test item can be assumed to be inhibitory and the test should be repeated using a lower concentration of test item - actual: 62% by day 14

4. The O2 uptake in the blank control should normally be 20-30 mg O2/L and should not be greater than 60 mg O2/L in 28 days. - actual: 19.6 mg O2/L

5. The pH value should be in the range of 6-8.5 - at the end of the test: pH blanks: 7.3; pH test item solution: 7.5 and 7.7; pH reference item solution: 7.6 and 7.5; pH toxicity control: 7.2

Applicant's summary and conclusion

Validity criteria fulfilled:
see: ''Any other information on results'' section
Interpretation of results:
readily biodegradable
In the Manometric Respirometry Test according to OECD 301F the test item was found to biodegrade (based on O2 consumption) for 85% within 10 days of incubation and, therefore, can be termed as readily biodegradable.
Executive summary:

In a test perfomed according to OECD 301F (Manometric Respirometry Test) and GLP, the test item biodegraded for 85% after 28 days. The pass level of 60% for ready biodegradability was reached within the 10 day window after attaining 10% degradation. It can therefore be termed as readily biodegradable.