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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
The mineral nutrient solution is directly prepared in the test vessels. The pH value of the mineral nutrient solution is checked and adjusted to pH 7,4 ± 0,2 if necessary. The titration of barium hydroxide solution is performed potentiometrically.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Sunflower oil, ester with sorbitol
EC Number:
EC Name:
Sunflower oil, ester with sorbitol
Cas Number:
Sunflower oil, ester with sorbitol
Specific details on test material used for the study:
Test item: Hostacerin SFO
Concentration of test item:S1: 53,2 mg/3I = 17,4 mg/l ; S2: 60,3 mg/3l = 20,1 mg/l
Application: direct weight
TOC: 71,0% (m/m)
ThCO2 in the test vessel: S1: 136,2mg ; S2: 157,0mg
Carbon content in the vessel: S1: 37,1 mg/3I = 12,4 mg/l ; S2: 42,8 mg/3l = 14,3 mg/l

Reference item: Sodium benzoate
Test concentration:R: 60,0 mg/3l = 20,0 mg/l
Application: direct weight
TOC: 58,3% (m/m)
ThC02 in the test vessel: R: 128,3 mg
Carbon content in the vessel: R: 35,0 mg/3l = 11,7 mg/l

Toxicity control
Concentration of test item: T: 57,5 mg/3l = 19,2 mg/l
Concentration of reference item: Tr: 60,0 mg/3l = 20,0 mg/l
Application: direct weight
ThC02 in the test vessel: T: 278,0 mg

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Activated sludge was aerated for 4 days to reduce endogenous respiration prior to use. On day 4 the loss of water due to evaporation was adjusted and a portion of approx. 1,5l was homogenised in a mixer at medium speed for 2 min.
Source :aeration tank of the municipal sewage treatment plant in Frankfurt-Niederrad, which is treating predominantly domestic sewage
Inoculum : activated sludge suspension (1,6 g/l dry substance)
Inoculum concentration : 18 ml/l
Bacterial count in test vessels : 104 cfu/ml
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
12.4 mg/L
Based on:
Initial conc.:
14.3 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Chemicals: All chemicals used are of analytical grade.
Test vessels: The test is performed in 5-l brown glass bottles equipped with gas inlet and gas outlet tube. The volume of test solution is 3 litres.
Mineral nutrient solution: The mineral nutrient solution is prepared according to guideline OECD 301 B.
Duration of the study: 28 days
Application of test item: once, at start of the test
Temperature: 22±2 °C

The necessary amounts of nutrient medium and inoculum are placed in each incubation vessel. The pH of the solution is checked, documented and adjusted to pH 7, 4 ± 0,2 if necessary. Afterwards the vessels are aerated with CO2-free air overnight to purge the system of carbon dioxide.
After addition of test item and reference item to the respective vessels the gas outlet tube of each vessel is connected to a series of three gas wash bottles, each containing 100 ml of barium hydroxide solution (c = 0,0125 mol/l).
The test is started by bubbling carbon dioxide-free air through the suspensions at a rate of 30-100 ml/min. The test suspensions are continuously stirred with magnetic stirrers.

The degradation is followed by C02 analysis at frequent intervals over a 28-day period. On the days of CO2 measurement the barium hydroxide bottle closest to the test vessel is disconnected. The remaining absorbers are moved one place closer to the test vessel and a new absorber containing 100 ml fresh 0,0125 mol/l barium hydroxide solution is placed at the far end of the series. If in the second absorber bottle a noticeable white deposit or precipitate of barium carbonate is observed, this bottle is also replaced by a fresh absorber bottle with 100 ml 0,0125 mol/l barium hydroxide solution.
The content of remaining barium hydroxide in the disconnected absorber bottles is titrated potentiometrically with 0,05 mol/l hydrochloric acid standard volumetric solution.
On day 28 the pH of the test suspensions is measured and documented. If the test item is water soluble 100 ml of each test suspension (S1, S2), inoculum control (C1, C2) and functional control (R) can be withdrawn for DOC analysis. 1 ml concentrated hydrochloric acid is added to each test vessel and the test vessels are aerated overnight to drive off the carbon dioxide present in the test suspensions. On day 29 the last analysis of evolved carbon dioxide is made.

The temperature in the test laboratory is continuously recorded with a thermo hygrograph during the test period.

The aeration of the test vessels is checked and documented each working day. Does the gas flow differ more than± 1 from the originally adjusted value, it is re-adjusted.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
A toxicity test, containing both the test item and the reference item, reached > 25 % biodegradation within 14 days and 84 % after 28 days. The test item is not inhibitory.

BOD5 / COD results

Results with reference substance:
Due to a leakage between day 1 and day 2 not the complete evolved carbon dioxide could be determined. Nevertheless the characteristics of the resulting biodegradation curve corresponds to a typical biodegradation curve of sodium benzoate with a less steep slope in the beginning. Therefore the validity criterion is regarded as valid.

Any other information on results incl. tables

      S1     S2
 Study day  Net sum [mg CO2/3l]  Degr [%]  Net sum [mg CO2/3l]

 Degr [%]

 2  13,8 10   12,3
 5  45,5 33   46,6 30 
 7  70,6 52   77,5 49 
 9  87,7 65   98,9 63 
 12  96,2 71   109,7  70
 16  105,4 78   122,4 78 
 21  111,3 82   132 84 
 28  117,6 87   141,2 90 

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The test item is readily biodegradable in the 10-d-window and after 28 days.
Executive summary:

The ready biodegradability of the test item was tested in the CO2 Evolution Test with non-adapted inoculum over a period of 28 days according to OECD Guideline for Testing of Chemicals 301 B. The test item in the concentration of 17 mg/l and 20 mg/l, respectively reached a degree of degradation of 86% and 90% after 28 days, respectively. The level of 10% degradation (begin of biodegradation) was reached after 2 days. The 60% level was reached after 8-9 days.

The test item must be regarded as readily biodegradable in the 10-d-window and after 28 days.

In order to check the validity of the test sodium benzoate in a concentration of 20 mg/l was used as reference item. The biodegradation of the reference item was not evaluated, because due to a leakage between day 1 and day 2 not the complete evolved carbon dioxide could be determined. In a toxicity test, containing both the test substance and the reference substance, the degradation reached 84%. The test item can be assumed to be not inhibitory. The validity criteria according to the guideline were fulfilled in the test.