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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:
1999-11-10 to 2000-01-05
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 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
according to guideline
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
according to guideline
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide
EC Number:
EC Name:
1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide
Cas Number:
Molecular formula:
1-(3,5-dihydroxyphenyl)-2-{[2-(4-hydroxyphenyl)-1-methylethyl]amino}ethanone hydrobromide
Test material form:
solid: particulate/powder

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):
activated sludge from a domestic waste water treatment plant supplied by sewage plant Groß-Zimmern, Germany
- Preparation of inoculum for exposure:
The activated sludge used for this study was washed by centrifugation and 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 suspension was weighed, dried and the ratio of wet 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 concentrationopen allclose all
Initial conc.:
3.26 mg/L
Based on:
test mat.
Initial conc.:
5.2 mg/L
Based on:
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, the test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was 8.6-8.7 mg/L at about 22°C.
- Test temperature: 22°C
- pH: 7.5 - 7.6
- Aeration of dilution water: yes

- Culturing apparatus: Karlsruher flasks of 250 ml volume with glass stopper
- 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: oxygen electrode (WTW Oxi 340)
- Test performed in closed system: yes

- Sampling frequency: Oxygen measurements were performed in duplicate on days 0, 2, 5, 7, 12, 14, 21 and 28 after the start of the test

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

Reference substance
Reference substance:

Results and discussion

% Degradation
other: based on ThOD (NH4)
Sampling time:
28 d
Details on results:
The residual oxygen concentration in the test flasks did not drop below 0.5 mg O2/L at any time. The difference of duplicate values for the degradation of the test substance at the plateau, at the end of the test and at the end of the 10-day window was less than 20%.

BOD5 / COD results

Results with reference substance:
The reference compound Aniline was sufficiently degraded to in the mean 72% after 14 days, and to 97 after 28 days of incubation. The percentage biodegradation of the reference compound confirms the suitability of the used activated sludge inoculum.

Any other information on results incl. tables

In the modified Closed Bottle test 3.26 mg/L of the test item were tested, corresponding to a ThOD of about 5.2 mgO2/L.

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

Percentage biodegradation (based on ThODNH4) at different time intervals during the exposure period of 28 days

 Treatment     Conc. [mg/L]     flask No.                       % biodegradation after n days of exposure
 2  5  7  12  14  21  28
 Test substance        3.26        1a  6  10  5  19  24  27  35
 1b  6  10  7  15  28  31  31
 mean  6  10  6  17  26  29  33
 Procedure Control (aniline)        2.0        2a  2  54  55  66  72  95  95
 2b  2  54  55  68  72  95  98
 mean  2  54  55  67  72  95  97
 Toxicity control      

 Test substance: 3.2 

Aniline: 2.12  

 4a  n. d.  n. d.  38  n. d.  52  63  65
 4b  n d.  n. d.  37  n. d.  52  63  65
 mean  n. d.  n. d.  38  n. d.  52  63  65

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The test item was not readily biodegradable under the conditions of the test.
According to the test guidelines the test substance can be assumed to be not inhibitory on the activated sludge micro organisms because degradation was > 25 % within 14 days.