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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:
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:
adopted July 17, 1992
according to guideline
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
December 1992
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,4,5-trimethoxycinnamic acid
EC Number:
EC Name:
3,4,5-trimethoxycinnamic acid
Cas Number:
Molecular formula:
3-(3,4,5-trimethoxyphenyl)acrylic acid
Test material form:
Details on test material:
Specific details on test material used for the study:
Batch 20031202
Expiry date: December 2005

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 Gro13-Zimmem, Ge1many
- Conditioning:
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 centri- fuged. 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, corre- sponding to 4 g d1y material per litre were mixed with test water (see 6.5) and then aerated until use. Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
15 mg/L
Based on:
Th0D(NH4) of 1.75 mg O/mg test item,
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium:
Reconstituted Water:
In deionised water analytical grade salts were added to prepare the following stock solutions:
a) 8.5 g KH2P04, 21.75 g K2HP04, 33.4 g Na2HP04 x 2 H20, 0.5 g NH4Cl filled up with deionised water to 1000 mL volUI1le
b) 22.5 g MgS04 x 7H20 filled up with deionised water to 1000 mL volume
c) 36.4 g CaCh x 2H20 filled up with deionised water to 1000 mLvolume
d) 0.25 g FeCIJ x 6H20 filled up with deionised water to 1000 mLvolume
In order to avoid preparation of the stock solution d) immediately before use, one drop of concentrated HCl per litre was added.
1 mL of the stock solutions a)- d) were combined and filled to a final volume of I000 mL with 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 8.5 to 8.7 mg!L at about 20 °C.

- Test temperature: 22.0 - 22.4 °C
- pH: 7.4 (measured at the start ofthe test)
- pH adjusted: no
- Continuous darkness: yes

- Karlsruher flasks of 250 mL volume with glass stoppers
- Number of culture flasks/concentration:
16 bottles containing the test item and inoculum
16 bottles containing the reference item Aniline and inoculum (procedure control)
16 bottles containing only inoculum (inoculum control)
10 bottles containing the test item. reference item Aniline and inoculum (toxicity control)
- Measuring equipment: 02-electrode (WfW Oxi 340, Wissenschaftlich Technische Werkstattcn GmbH, Weilheim, Germany) under constant stirring.

Oxygen measurements were performed in duplicate on days 0, 3, 5, 7, 12, 14,21 and 28 (measurements of the toxicity con- trol were done only on days 0, 7, 14, 21 and 28).

Reference substance
Reference substance:

Results and discussion

Test performance:
The reference item Aniline was sufficiently degraded to a mean of 81 % after 14 days, and to a mean of 104 % after 28 days of incubation.
The percentage biodegradation of the reference item con- firms the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item Aniline, a mean of 76 % biodegradation was noted within 14 days and was within this range up to 28 days of incubation.
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.
% Degradationopen allclose all
Key result
% degradation (O2 consumption)
Sampling time:
12 d
Key result
% degradation (O2 consumption)
Sampling time:
21 d

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The percentage biodegradation exceeded 60 % within the 10-day window (between day 13, extrapolated, and day 14). The test item can therefore be considered to be readily