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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2011-06-15 to 2011-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Analytical purity: 100.5 %
- Lot/batch No.: 10191-AKm
- Expiry date: 2013-06-16
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary on 15 June 2011.
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution 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 5 g dry material per litre was mixed with reconstituted water (see above) and then aerated until use (in this study 100 mL inoculum was prepared). Before use the sludge was filtered through cotton wool. The pH of the activated sludge inoculum was checked after preparation and before use. The pH of the activated sludge inoculum after preparation was 6.91. pH adjustment before use was not performed.
- Concentration of sludge: 5 g dw/L
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to the guideline
- Test temperature: 22 ± 2 °C
- pH: prior study 7.26
- pH adjusted: yes
- Aeration of dilution water: aeration of dilution water until use
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Winkler bottles with glass stoppers
- Number of culture flasks/concentration:
10 (+2 reserve) bottles containing the test item and inoculum
10 (+2 reserve) bottles containing the reference item and inoculum (procedure control)
10 (+2 reserve) bottles containing only inoculum (inoculum control)
10 (+2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)
- Method used to create aerobic conditions: aeration system
- Measuring equipment: Complex measuring system with O2 electrode and pH electrode and built in temperature probe Aeration system, Incubator with thermometer, Balance, Centrifuge, Moisture analyzer.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only filtered inoculum was added to the aqueous test medium.
- Toxicity control: Adequate amount of Sika Hardener MH and reference item stock solution was mixed into the aqueous test medium corresponding to the investigated 2.0 mg/L test item concentration [chosen based on the theoretical oxygen demand (ThODNO3 of 6.0 mg O2/L) of the test item] and to 3.6 mg/L concentration of the reference item (ThODNH4 of 6.012 mg O2/L).
- Procedure Control: Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), at the start of the test a stock solution of Sodium benzoate was mixed into the aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L).
Reference substance:
benzoic acid, sodium salt
Test performance:
The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
Key result
Parameter:
% degradation (O2 consumption)
Value:
2.9
Sampling time:
28 d
Details on results:
Because of the N-containing test item, the oxidised nitrogen (nitrate and nitrite) concentrations were determined after each oxygen measurement with photometric method using nitrite and nitrate cell tests. The LOQ of the measurements was 0.03 mg NO2/mL and 0.4 mg NO3/mL, respectively. The measured quantities of nitrite and nitrate were below the LOQ (or were not detectable) in the measured samples throughout the study. Therefore, the BOD values were not corrected for nitrification, because the quantities of the oxidised nitrogen (nitrite and nitrate) were below of the LOQ throughout the study.
Under the test conditions the percentage biodegradation of Sika Hardener MH reached a mean of 2.9 % after 28 days based on ThODNO3. (The highest percentage biodegradation value, 8.8 % of Sika Hardener MH was observed on the 21st day based on ThODNO3.). The test item can be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNO3.
Key result
Parameter:
BOD5
Value:
> 0 - <= 0.15 mg O2/g test mat.
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 60.5 % after 14 days, and to a mean of 76.3 % after 28 days of incubation, based on ThODNH4. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration
[mg/L]

Flask
No.

Percent of biodegradation after n days of exposure

7

14

21

28

Test Item

2.0

1a

7.6

3.7

10.1

3.4

1b

2.3

1.0

7.6

2.4

 

Mean

4.9

2.3

8.8

2.9

Reference item

3.6

2a

63.1

59.8

63.3

76.6

2b

75.3

61.2

65.4

75.9

 

Mean

69.2

60.5

64.3

76.3

Toxicity control

Test item: 2.0
Reference item: 3.6

4a

28.5

30.9

35.8

35.9

4b

29.7

31.0

36.0

36.4

Mean

29.1

31.0

35.9

36.2

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item can be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNO3.
Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item Sika Hardener MH. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. Under the test conditions the percentage biodegradation of Sika Hardener MH reached a mean of 2.9 % after 28 days based on ThODNO3, (the highest percentage biodegradation value, 8.8 % of Sika Hardener MH was observed on the 21st day based on ThODNO3.). Therefore. the test item can be considered to be not ready biodegradable. The reference item Sodium benzoate was sufficiently degraded to a mean of 60.5 % after 14 days (69.2 % degradation was observed already on the 7th day of the test), and to a mean of 76.3 % after 28 days of incubation, based on ThODNH4, thus, confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 31.0 % biodegradation was noted within 14 days and 36.2 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Description of key information

The ready biodegradability of the test item was assessed over a 28 day period in a closed bottle test (EU method C 4-E). The test material attained up to 2.9 % biodegradation after 28 days whereas the reference substance (Sodium benzoate) was 60.5 % degraded 28 days. These results indicate that the test item cannot be classified readily biodegradable under conditions of the test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The purpose of this study was to determine the ready biodegradability of the test item Sika Hardener MH. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. Under the test conditions the percentage biodegradation of Sika Hardener MH reached a mean of 2.9 % after 28 days based on ThODNO3, (the highest percentage biodegradation value, 8.8 % of Sika Hardener MH was observed on the 21st day based on ThODNO3.). Therefore. the test item can be considered to be not ready biodegradable. The reference item Sodium benzoate was sufficiently degraded to a mean of 60.5 % after 14 days (69.2 % degradation was observed already on the 7th day of the test), and to a mean of 76.3 % after 28 days of incubation, based on ThODNH4, thus, confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 31.0 % biodegradation was noted within 14 days and 36.2 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).