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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28/July/2001 - 25/Aug/2001
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The work reported was performed carefully, the results reasonable, and the conclusions conservative. Study was conducted in accordance with OECD guidance. Results for test and reference substances met criteria for a valid test result.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report: T-7145
- Physical state: Colorless liquid
- Analytical purity: 99.89%
- Stability under test conditions: Stable

Study design

Oxygen conditions:
Inoculum or test system:
other: Secondary effluent from domestic sewage treatment plant
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Domestic sewage treatment plant of Yokohama Research Center, Mitsubishi Chemical Corporation, collected 28 July 1999.
- Laboratory culture: BOD measurement began same day as collection
- Pretreatment: None
- Concentration of sludge: 500 microliter/L
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
9.13 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: Standard dilution water described in OECD guideline for testing of
chemicals 301D was used as mineral medium.
- Test temperature: 20 ± 1 °C
- pH: 6.8-7.1
- pH adjusted: No
- CEC (meq/100 g): Not reported
- Aeration of dilution water: Before test
- Suspended solids concentration: Not reported
- Continuous darkness: Yes
- Culturing apparatus: TAITEC, Model M-210 incubator
- Number of culture flasks/concentration: 14 each for test substance and abiotic control, 11 for blank, 10 for reference substance.
- Method used to create aerobic conditions: Mineral medium and ultrapure water were saturated with oxygen.
- Measuring equipment: Orion Research, Model 720A DO meter
- Test performed in closed vessels due to significant volatility of test substance: yes, closed BOD bottle with sealing water around the glass stopper.
- Sampling frequency: Weekly (DO measurement), on Day 0 and Day 28 (pH and extraction of residual test substance)
- Sampling method: Not reported
- Sterility check if applicable: Not reported
- Sample storage before analysis: Not reported
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: No
Reference substance
Reference substance:

Results and discussion

Preliminary study:
% Degradation
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
REMARKS for section "% Degradation of test substance":
ThOD for reference substance (aniline) calculated using:
C6H7N + 7O2 -> 6CO2 + 2H20 + NH3, 2.41 mg O2/mg reference substance (does not consider nitrification)

ThOD for test substance calculated according to MITI method using:
C9H5OF15 + (39/4)O2 -> 9CO2 + (5/2)H2O + 15F, 0.75 mg O2/mg test substance

Study report included ThOD for test substance calculated according to OECD method using:
C9H5OF15 + 6O2 -> 9CO2 + 5H2O + 15HF, 0.46 mg O2/mg test substance

Calculations were performed using the MITI ThOD value. From the degradability results based on the BOD and the residual test substance concentration, it is concluded that the test substance was not readily biodegradable and was not transformed under the conditions of the Closed Bottle Test.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: Solubility reported as <1 ppm, test substance incorporated in to medium at 9.13 ppm
BOD v. Time plot - see attachment 1

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
CAS# 297730-93-9 was not readily biodegradable under test conditions.
Executive summary:

The ready biodegradability of CAS# 297730-93-9 was studied in a 28-day closed bottle test according to OECD method 301D.  Secondary effluent of a domestic sewage treatment plant was used as a source of inoculum. BOD bottles were filled with oxygen-saturated medium and spiked with either CAS# 297730-93-9 at 9.13 mg/L (ThOD, 0.75 mg O2/mg), aniline at 2.00 mg/L (reference substance, ThOD, 2.41 mg O2/mg), or no additional substance (inoculum blank).  Additional BOD bottles were filled with oxygen-saturated ultrapure water and spiked with CAS# 297730-93-9 at 9.13 mg/L (abiotic control). Dissolved oxygen concentration was measured at seven-day intervals from test initiation (Day 0) to termination (Day 28).  pH was measured in bottles after DO measurements were taken on Days 0 and 28 only. Additional BOD bottles were extracted with chloroform for gas chromatographic analysis (GC) of CAS# 297730-93-9 on Days 0 and 28 only (one inoculum blank on day 0, two each of CAS# 297730-93-9 and abiotic controls on Days 0 and 28). The 28-day biodegradation according to oxygen depletion (100% * BOD/ThOD) was 1%. The 28-day depletion of CAS# 297730-93-9 was 0-1% by GC.  The reference substance was completely degraded on Day 28. Based on these data, CAS# 297730-93-9 was not readily biodegradable was not transformed under the conditions of the Closed Bottle Test.


This study is classified as acceptable and satisfies the guideline requirements for test method OECD301, closed bottle ready biodegradability test.


Results Synopsis


Test Organism Age: less than one day

Test Type: Closed bottle


Percent degradability: 1%                     95% C.I.:  not applicable

Endpoint(s) Effected: