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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:
13 September - 17 October 2000
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 301 D) and in compliance with GLP.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
according to
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
according to
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): TH 1165 I; (4-Methoxyphenyl)-aceton
- Physical state: yellow liquid
- Analytical purity: 99.01 %
- Purity test date: 6. March 2000
- Lot/batch No.: 14
- Expiration date of the lot/batch: can be requested from the sponsor
- Stability under test conditions: can be requested from the sponsor
- Storage condition of test material: in original container, at room temperature, in the dark

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): supplied by sewage plant Groß-Zimmern, Germany

The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water and then areated until use. Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2.58 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: grade salts were added to deionised water to give stock solutions. Stock solutions were diluted in deionised water, aerated for 20 minutes and allowed to stand for 20 hrs at the test temperature. Dissolved oxygen concentration was about 8.7 - 8.9 mg/L prior to starting the test.
- Test temperature: 22°C
- pH: 7.6

- Culturing apparatus: 250 ml flasks with glass stoppers
- Number of culture flasks/concentration: 16 bottles containing the test substance and inoculum; 16 bottles containing the reference compound Aniline and Inoculum (procedure control); 16 bottles containing only inoculum (inoculum control); 10 bottles containing the test substance, reference compound Aniline and inoculum (toxicity control)
- Method used to create aerobic conditions: inoculum and medium solutions were sufficiently aerated prior to the start of the test.
- Measuring equipment: O2 electrode
- Test performed in closed vessels: yes

- Sampling frequency: oxygen was measured at days 0, 2, 5, 7, 12, 14, 21 and 28 after the start of the test.
- Sampling method: no sampling was performed for oxygen analysis, the oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode.
- Sterility check if applicable: no
- Sample storage before analysis: no

- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance
Reference substance:

Results and discussion

% Degradation
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
The residual oxygen concentration did not drop below 0.5 mg O2/L at any time. The difference in duplicate samples was less than 20%.

BOD5 / COD results

Results with reference substance:
The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge micro organisms because degradation was >25% within 14 days.

Any other information on results incl. tables

Percentage biodegradation at different time intervals during the exposure period of 28 days

 Treatment     Conc. [mg/L]     flask No.                       % biodegradation after n days of exposure
 7  14  16  19  21  24  28
 Test item        2.58  1a  1  17  21  34  47  61  75
   1b  1  19  24  32  44  60  73
   mean  1  18  22  33  46  60  74
 Procedure Control (aniline)    2.00  2a  76  86  88  90  92  93  110
   2b  72  86  90  92  90  87  114
   mean  74  86  89  91  91  90  112
 Toxicity Control        TI: 2.58  4a  35  49  n.d.  n.d.  73  n.d.  70
 Aniline: 2.08  4b  36  50  n.d.  n.d.  74  n.d.  71
   mean  35  50  n.d.  n.d.  73  n.d.  70

n.d.=not determined; TI= test item

BOD = (mg O2/L of test item and/or Aniline - mg O2/L of inoculum control)/(mg test item and/or Aniline/L in flask)

%biodegradation = (BOD * 100%)/ (ThOD(NH4) of Aniline and/or test item)

where: ThOD(NH4) of test item = 2.34 mg O2/mg test item

ThOD(NH4) of Aniline = 2.41 mg/O2 mg Aniline

The percentage biodegradation at the end of the 10 -day window was detrmined to be in the mean 46% and degreased to 74 % until the end of the test.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
other: not readily biodegradable
Since the percentage biodegradation was below 60% within the 10 day window, the test item can not considered to be ready biodegraable.