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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:
08 December 2016 - 05 January 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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
other: Ministry of environmental protection of the people's Republic of China, The Guideline for Testing of Chemicals - Degradation and Accumulation, 2013, 301F Ready Biodegradability: Manometric Respirometry Test
Version / remarks:
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
C21 H30 N6 O6
Constituent 2
Chemical structure
Reference substance name:
2-methylpropyl N-(5-isocyanatopentyl)-N-(5-isocyanatopentylcarbamoyl)carbamate
Molecular formula:
C18 H30 N4 O5
2-methylpropyl N-(5-isocyanatopentyl)-N-(5-isocyanatopentylcarbamoyl)carbamate
Test material form:
liquid: viscous
Details on test material:
Appearance: colourless or light yellow viscous liquid
Storage conditions: at room temperature, container tightly closed, flushed with nitrogen.

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of sludge: aeration tank of the Long Hu Wastewater Treatment Plant, Shanghai (collected on 8 December 2016, experimental start date).
- Storage conditions: the sludge was kept under aeration until used
- Storage length: the sludge was used on the day of collection
- Preparation of inoculum for exposure: not applicable
- Pretreatment: the sludge was washed with mineral medium and, after centrifugation, the supernatant was decanted. This procedure was repeated three times (4000 rpm, 4°C, centrifuge 20 minutes). Then, 1.207 g of the concentrated sludge was weighed, dried at 105°C for 1 hour. After drying, the dry weight content was determined to be 7.37%.
- Initial cell/biomass concentration: 3 g suspended solids/L (40.7 g of concentrated sludge suspended in 1000 mL mineral medium)
- Final concentration of sludge in the test medium: 30 mg/L

Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
54.41 mg/L
Based on:
mean of 2 replicates
Initial conc.:
35.1 mg/L
Based on:
test mat.
mean of 2 replicates
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: the medium was according to OECD 301F.
- Test temperature: 22.0 - 22.6°C
- pH: 6.72 - 7.39
- pH adjusted: no
- Aeration of dilution water: not indicated
- Suspended solids concentration: 2.5 mL of the 3 g suspended solids/L solution was added to the test item with 247.5 mL of test medium, making up a final suspended solids concentration in the test solution of 30 mg/L.
- Continuous darkness: yes

- Culturing apparatus: brown BOD bottles, airtight, volume: 500 mL
- Number of culture flasks/concentration: 2, plus one used for the pH analysis
- Method used to create aerobic conditions: not indicated
- Measuring equipment: BOD respirometer: WTW, OxiTop 110C (Weilheim, Germany)
- Test performed in open system: no
- Details of trap for CO2: 2 sodium hydroxide pellets were placed in rubber sleeves which were inserted into the flasks. The sodium hydroxide absorbed the generated CO2.
- Other: the test solutions were continuously stirred.

- Sampling frequency: once per 112 min during the entire exposure period (i.e., 28 days).
- Sampling method: the pressure drop (caused by absorption of CO2 by sodium hydroxide) was measured by an electrode type manometer.

- Inoculum blank: yes, 2 replicates
- Toxicity control: yes, 1 replicate
- Other: a procedure control (2 replicates) containing reference substance, sludge and mineral medium.
- For each of the above, one additional flask was used for the zero-time pH analysis.

STATISTICAL METHODS: no statistical analysis methods were reported.
Reference substance
Reference substance:
benzoic acid, sodium salt
Purity: 99%

Results and discussion

% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: mean of 2 bottles
Details on results:
- Individual bottles with test substance showed biodegration percentages of -1% (considered as 0%) and 2%, resulting in a mean biodegradation of 1%.
- Biodegradation of the toxicity control after 14 days of exposure was 67%, indicating that the test substance had no toxic or inhibitory effect on the acitivity of the microbial inoculum.

BOD5 / COD results

Results with reference substance:
84% after 14 days of exposure (n=2)

Applicant's summary and conclusion

Validity criteria fulfilled:
See 'Overall remarks, attachments'
Interpretation of results:
not readily biodegradable
The test substance showed a mean biodegradation of 1% after 28 days. Based on this result, STABiO D-376N is not readily biodegradable.
Executive summary:

The biodegradability of STABiO D-376N was determined in a Manometric Respirometry Test, performed according to OECD test guideline 301F and GLP principles. The substance was tested in duplicate for a period of 28 days at a nominal concentration of 35 mg/L (mean of replicates), corresponding to 54.41 mg ThOD/L. Two inoculum blanks, two reference controls and one toxicity control were also included. Activated sludge from a domestic waste water treatment plant was used as inoculum (30 mg/L) and the reference substance was sodium benzoate (100 mg/L, corresponding to 167 mg ThOD/L). Over the 28 -day exposure period, the pressure drop, as a result of O2 consumption and resulting CO2 production, which was absorbed by NaOH, was measured every 112 minutes by a respirometer. These data were used to calculate the biodegradability of the test substance, reference substance and the toxicity control. At the end of the test, the biochemical oxygen demand (BOD) of the test substance was comparable to the BOD of blank controls and the mean biodegradation percentage of the test substance at day 28 was 1% (no 10 -day window). Based on these results, STABiO is considered not readily biodegradable. Over a period of 14 days, the reference substance was biodegraded for 84%. The toxicity control was biodegraded for 67% indicating that the test substance was not toxic or inhibiting to the microbial inoculum. All acceptability criteria were met and the test was considered to be valid.