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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:
11 October 2016 - 9 November 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:
17 July 1992
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 3
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Dihydrogen oxide
impurity 1
Reference substance name:
Unknown impurities
Unknown impurities
Test material form:
Details on test material:
- Appearance: Slightly yellow gel
- Storage conditions: At room temperature; container flushed with nitrogen
Specific details on test material used for the study:
Theoretical oxygen demand: 2.857 mg O2/mg, anticipating nitrification
Water solubility: = 5.3 g/L

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Non-adapted activated sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant; sampling: on 6 October 2016
- Storage conditions and period: sludge was pre-conditioned for 6 days (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 (60 mg dry matter/L).
- Initial cell/biomass concentration: 2 g dry matter/L
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
97.1 mg/L
Based on:
Initial conc.:
34 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 (according to OECD 301) prepared with ultrapure water (conductivity: <1.5 µS/cm; DOC: <0.5 mg/l)
- Test temperature: 22 ±2 °C
- pH adjusted: yes, at the start of the test the pH was adjust if necessary to 7.4 (+/- 0.2) with NaOH or HCl (except in flask C; and in flask T and X only if the test item is soluble).
- pH at the end of the exposure period: blank control: 7.3, procedure control: 7.5, test suspensions: 7.9 and 7.4, toxicity control: 7.6.
- Suspended solids concentration: 30 mg dry matter/L
- Continuous darkness: not indicated

- Culturing apparatus: 510 mL glass bottles, closed with manometric BOD measuring devices, fill volume: 200 mL. Pellets of NaOH were added to absorb the produced CO2 from the headspace.
- Number of culture flasks/concentration: 2
- Measuring method: 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)
- Measuring equipment: Dissolved organic carbon (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.

- Inoculum blank: yes, 2 replicates
- Abiotic sterile control: yes, containing test item, ultra-pure water and 0.04 mM HgCl2 as sterilizing agent to prevent microbial decomposition, 1 replicate
- Toxicity control: yes, 2 replicates
- Other: procedure control containing inoculum, test medium and sodium benzoate as ready biodegradable reference item, 1 replicate

STATISTICAL METHODS: no statistical analysis was performed.
Reference substance
Reference substance:
benzoic acid, sodium salt
Batch: 15D280007 (expiring 31 October 2019); Purity: 99.8%; 100 mg ThOD/L

Results and discussion

% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: Actual value for mean degradation of replicates: -4.2%
Details on results:
No biodegradability of 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) based on O2 consumption was observed after 28 days as compared to the theoretical oxygen demand (ThOD).

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:
23.8 and 3.06 for the test units
<0.5 and 0.90 for the blank control
35.1 and 1.16 for the procedure control.
The total elimination based on dissolved organic carbon (DOC) measurements reached 91% for the test item and 99% for sodium benzoate, respectively.

The procedure control sodium benzoate reached 98% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

At the applied initial test concentration of 37.0 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. Although the toxicity control exceeded the 25% limit within 14 days as required by the guideline, an initial lag phase of 3 days was observed before degradation occurred. This was presumably due to an initial inhibitory effect of the test item on the microbial population and is not considered to have an effect on the final results.

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:
98% biodegradation after 14 days.

Any other information on results incl. tables

Validity criteria

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: 0% at test end (day 28)

2. The percentage of degradation of the reference item must reach the pass level of 60% by day 14 - actual: 98% 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: 37.6% by day 14

4. The O2 uptake in the blank control should normally be approximately 20-30 mg O2/L and should not be greater than 60 mg O2/L in 28 days. - actual: 14.4 mg O2/L

5. The pH value should be in the range of 6-8.5. - at test end: pH blanks - 7.3; pH test item solution - 7.9 and 7.4; pH reference item solution - 7.5 and 7.6; pH toxicity control - 7.6

All validity criteria were fulfilled.

Applicant's summary and conclusion

Validity criteria fulfilled:
See 'any other information on results'.
Interpretation of results:
not readily biodegradable
In the Manometric Respirometry Test according to OECD 301F the test item did not biodegrade (based on O2 consumption) after 28 days of incubation and, therefore, cannot be termed as readily biodegradable.