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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:
20 June - 19 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
EC 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
June 2012
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: A sample of activated sludge was obtained from Worlingworth sewage treatment works (Suffolk, UK), which treats predominantly domestic waste.
- Laboratory culture: At the time of collection, the sludge was sieved (1 mm2 mesh sieve) then transported to the laboratory and left to stand for approximately 30 minutes to allow the sewage solids to settle. A portion of the supernatant was removed and the sludge aerated until required.
- Pretreatment: The concentration of suspended solids in a homogenised sample was determined before the start of the test. Aliquots (10 mL) of the sludge were filtered through dried and preweighed Whatman GF/C filters, which were then dried again at approximately 105°C for one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the sludge was then determined and the volume required to give a solids level of 30 mg/L in test cultures was calculated. This was added to bottles one day before test initiation to allow a period of ageing.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
ThOD
Remarks:
mg O2/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral salts medium (MSM) for the test was prepared by establishing 10 mL of stock solution 1 and 1 mL of solutions 2, 3 and 4 in each litre of water required for the test.
* Stock 1:
Potassium dihydrogen phosphate: 8.50 g/L
di-Potassium hydrogen phosphate: 21.75 g/L
di-Sodium monohydrogen phosphate dihydrate: 33.40 g/L
Ammonium chloride: 0.5 g/L
* Stock 2:
Magnesium sulphate heptahydrate: 22.50 g/L
* Stock 3:
Calcium chloride dihydrate: 36.40 g/L
* Stock 4:
Iron (III) chloride hexahydrate: 0.25 g/L
Minor variations in the weights taken are not considered to be significant.
- Test temperature: 20.1 to 24.8℃ (nominal 22 ± 2ºC)
- pH: The pH of test mixtures ranged between 7.47 and 7.52 at the start of the test and 7.47 and 7.64 at the end.
- pH adjusted: yes

TEST SYSTEM
- Culturing apparatus: amber glass culture bottles (500 mL)
- Oxygen consumption measurements: Co-ordinated Environmental Services (CES) Ltd automated respirometer
- Number of culture flasks/concentration: 2 blank-control; 1 reference; 2 test; 1 inhibition assay; 1 ATU control; 1 ATU test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculated MSM alone

TEST DESIGN
One day before test initiation, a volume (5 litres) of mineral salts medium (MSM) was prepared and the pH determined and adjusted to 7.6 with 5M HCl. Appropriate volumes of the MSM were then added to eight amber glass culture bottles (500 mL) and inoculated with activated sludge. Magnetic stirrer bars were added to the cultures and each bottle was fitted with an electrolytic cell assembly (containing the electrolyte, 1M copper sulfate solution, and the CO2 absorber, 5 mL of 2M potassium hydroxide) and connected to the respirometer. A magnetic stirrer was set to give a vortex in each test mixture and the instrument was initiated.

PREPARATION OF TEST VESSELS
At test initiation (Day 0), the test substance (nominally 14.1 mg; corrected for a purity of 94.2%) was weighed into four 25 mL volumetric flasks containing MSM; the preparations were treated with ultrasound for 15 minutes. The preparations were made up to volume with MSM and the contents then added to the appropriate culture bottle, followed by 2 x 25 mL rinses of MSM (75 mL in total) to give a final nominal concentration of 50 mgO2/L or 25 mgO2/500 mL.
In order to ensure similarity in the preparation of all mixtures, 75 mL of MSM alone were added to the control and reference cultures.
Sodium benzoate (20 mL) was added as an aqueous stock solution (0.750 g/L) in MSM to the reference and inhibition assay cultures to give a final nominal concentration of 25 mgO2/500 mL or 50 mgO2/L.
A stock solution of allylthiourea (ATU) was prepared in MSM at a nominal concentration of 1.16 g/L. An aliquot (5 mL) of this stock solution was added to one culture containing inoculated MSM alone and to one containing the test substance.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
St. dev.:
0
Sampling time:
28 d
Details on results:
Test substance: C21H14N2O4 MW = 358.35
ThOD ammonia: 1.88 mgO2/mg
ThOD nitrite: 2.14 mgO2/mg
ThOD nitrate: 2.23 mgO2/mg

The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 16.14 mgO2/500 mL or 65% of the ThOD (25 mgO2/500 mL) after 3 days of incubation and 21.02 mgO2/500 mL or 84% by Day 28. In the presence of the test substance, degradation of sodium benzoate had achieved 62% by Day 6. Cumulative levels of oxygen consumption by the controls after 28 days (19.99 and 19.36 mgO2/500 mL, equivalent to 39.98 and 38.72 mgO2/L) were considered to be acceptable for this assay system. These results confirm that the test substance was not inhibitory to the activity of the microbial inoculum and that the test was valid.
Oxygen consumption by the control mixture containing Allylthiourea (ATU) was equivalent to 15.61 mgO2/L on Day 28 of the test. By the end of the test, there was no oxygen consumption measured in the ATU treated test mixture above that measured in the ATU treated control.
There was no oxygen consumption in mixtures containing test substance above that of the control mixture during the test. Substances are considered to be readily biodegradable in this type of test if oxygen consumption is equal to or greater than 60% of the ThOD of the mixtures within ten days of the consumption achieving 10%. Therefore, the test substance was not considered to be readily biodegradable under the conditions of this test.
Results with reference substance:
Sodium benzoate: C7H5O2Na MW = 144 ThOD: 1.67 mgO2/mg. Degradation of sodium benzoate (65% of its ThOD after 3 days)

Day

Control; cumulative oxygen demands (mgO2)

BMI (50 mgO2/L); cumulative oxygen demands (mgO2)

BMI (50 mgO2/L); blank corrected (mgO2)

1

1.58

0.89

0

2

2.66

1.52

0

3

3.43

1.97

0

4

4.22

2.49

0

5

4.81

2.85

0

6

5.06

2.85

0

7

5.51

3.01

0

8

6.62

3.99

0

9

7.58

4.89

0

10

8.53

5.66

0

11

9.07

6.01

0

12

9.28

6.22

0

13

9.51

6.47

0

14

9.85

6.80

0

15

10.59

7.51

0

16

11.12

7.99

0

17

11.57

8.43

0

18

11.99

8.82

0

19

12.41

9.24

0

20

12.95

9.76

0

21

13.51

10.30

0

22

13.61

10.41

0

23

14.03

10.78

0

24

14.53

11.28

0

25

14.97

11.72

0

26

14.97

11.72

0

27

15.01

11.72

0

28

15.61

12.30

0

Validity criteria fulfilled:
yes
Remarks:
See 'overall remarks' for details on validity criteria.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance was not considered to be readily biodegradable under the conditions of this test.
Executive summary:

The 'ready' biodegradability of the test item was investigated in the Manometric Respirometry Test, according to OECD Guideline 301 F, EC 440/2008 C.4 -D, and GLP principles. A nominal test concentration of 50 mg O2/L ThOD was used. There was no oxygen consumption and no biodegradation in two replicates after 28 days. The pass criterion for ready biodegradation (60% biodegradation within a 10 -day window) was not met. Therefore, the test item is considered not readily biodegradable. The test item was not inhibitory to the activity of the microbial inoculum.

The test met all validity criteria, therefore the study is considered to be reliable without restrictions.

Description of key information

BMI was found to be not readily biodegradable in a OECD 301F Ready Biodegradability, Manometric Respirometry Test.

Key value for chemical safety assessment

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

Additional information

The 'ready' biodegradability of the test item was investigated in the Manometric Respirometry Test, according to OECD Guideline 301 F, EC 440/2008 C.4 -D, and GLP principles. A nominal test concentration of 50 mg O2/L ThOD was used. There was no oxygen consumption and no biodegradation in two replicates after 28 days. The pass criterion for ready biodegradation (60% biodegradation within a 10 -day window) was not met. Therefore, the test item is considered not readily biodegradable. The test item was not inhibitory to the activity of the microbial inoculum.

The test met all validity criteria, therefore the study is considered to be reliable without restrictions.