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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:
26 May 2016 to 23 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The respective amount of the test material was weighed directly to reach the required test material concentration of 1.9 mg/L. The chosen test material concentration was based on the measured chemical oxygen demand (COD): 3.24 ± 0.035 mg O2/mg test material (calculated according to the equation given in the guidelines) and on the performed14-day preliminary test.
Oxygen conditions:
anaerobic
Inoculum or test system:
sewage, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Sewage plant for domestic sewage in Veszprém, Hungary.
- Conditioning: The secondary effluent used for this study was allowed to settle for an hour, then the decanted effluent was aerated until use (not later than an hour).
Duration of test (contact time):
28 d
Initial conc.:
1.9 mg/L
Based on:
test mat.
Initial conc.:
6.156 other: mg O2/ mg test material
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Reconstituted water
Stock solutions:
a) Solution: KH2PO4 2.125 g, K2HPO4 5.4375 g, Na2HPO4 x 12H2O 16.795 g, NH4Cl 0.125 g and Deionised water ad 250 mL.
b) Solution: MgSO4 x 7 H2O 5.625 g and Deionised water ad 250 mL.
c) Solution: CaCl2 x 2 H2O 9.10 g and Deionised water ad 250 mL.
d) Solution: FeCl3 x 6 H2O 0.25 g and Deionised water ad 1 000 mL.
- Adequate amount of the stock solutions a) - d) were combined and filled up with deionised water to the appropriate final volume (ratio of composition referring to 1 mL of the stock solutions a) - d) filled up with deionised water to a final volume of 1 000 mL according to the guideline). 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 8.8 mg/L at about 22 °C.
- Test temperature: 20.7 - 23.5 °C. Temperature was measured continuously and registered on weekdays.
- pH: 7.42
- pH adjusted: No
- Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: BOD bottles (300 mL) with special neck and glass stoppers.
- Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.
- Number of culture flasks: 10 bottles containing the test material and inoculum, 10 bottles containing the reference material and inoculum (procedure control), 10 bottles containing only inoculum (inoculum control) and 10 bottles containing the test material, reference material and inoculum (toxicity control).
- Measuring equipment: The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28. The COD (chemical oxygen demand) of the test material was determined in the analytical department of the test facility using Lovibond® COD Measuring System.
- Based on the measured chemical oxygen demand (COD) of 3.24 mg O2/mg test material, 6.4 mg of test material was thoroughly mixed into 3.38 litres of aqueous test medium (corresponding to 1.9 mg/L test item, with a COD of about 6.156 mg O2/L).

CONTROL AND BLANK SYSTEM
- Inoculum control: Flasks 3a and 3b: Only filtered inoculum was added to 3.40 litres of aqueous test medium.
- Procedure control: Sodium benzoate flasks 2a and 2b. Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg), stock solution (360 mg/L) corresponding to 12.240 mg of Sodium benzoate was mixed into 3.40 litres of aqueous test medium (corresponding to 3.6 mg/L reference material, respectively a ThODNH4 of about 6.012 mg O2/L).
- Toxicity control: Flasks 4a and 4b. The Test Material (6.4 mg) and reference material stock solution (360 mg/L) (33.8 mL) were mixed into 3.38 litres of aqueous test medium corresponding to 1.9 mg/L test material (COD of 6.156 mg O2/L) and 3.6 mg/L reference material (ThODNH4 of 6.012 mg O2/L).

CALCULATION OF RESULTS
- Calculation of BOD:
The BOD (mg O2 per mg test material) expected after each period was calculated as follows:

(mg O2/L of T.m. and/or R.m. - mg O2/L of i.control ) / (mg T.m. and/or R.m./L in flask)

where:
T.m. = test material
R.m. = reference material,
i.control = inoculum control

- Calculation of Biodegradation %:
The percentage biodegradation of the test material and of the reference material was calculated as follows:

[(BOD(mg O2/mg T.m. or R.m.)) / (COD (mg O2/mg T.m.) or ThODNH4 (mg O2/mg R.m.))] x100

where:
T.m. = test material
R.m. = reference material
i.control = inoculum control
Reference substance:
benzoic acid, sodium salt
Test performance:
- In the toxicity control containing both, the test material and the reference material Sodium benzoate, a mean of 35.2 % biodegradation was noted within 14 days and 38.9 % biodegradation after 28 days of incubation. According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 1.9 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.
- The study met the validity criteria.
Key result
Parameter:
% degradation (O2 consumption)
Value:
3.2
Sampling time:
28 d
Details on results:
- The percentage biodegradation of the test material reached a mean of 3.2 % after 28 days based on the measured COD of the test material. Therefore the test material is considered not readily biodegradable.
- According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.
Results with reference substance:
- The reference material Sodium benzoate was sufficiently degraded to a mean of 78.3 % after 14 days, and to a mean of 90.0 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

Table 1: Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration (mg/L)

Flask No.

mg O2/L after n days of exposure

0

7

14

21

28

Test material

1.9

1a

8.5

8.0

7.7

7.7

7.6

1b

8.5

7.8

7.6

7.6

7.4

mean

8.50

7.90

7.65

7.65

7.50

Reference material

3.6

2a

8.5

4.2

3.2

2.7

2.4

2b

8.6

4.0

3.0

2.6

2.3

mean

8.55

4.10

3.10

2.65

2.35

Inoculum control

-

3a

8.5

8.1

7.8

7.9

7.8

3b

8.6

8.0

7.8

7.8

7.7

mean

8.55

8.05

7.80

7.85

7.75

Toxicity control

Test material: 1.9, Reference material: 3.6

4a

8.5

3.7

3.0

2.7

2.5

4b

8.5

3.7

3.0

2.6

2.4

mean

8.50

3.70

3.0

2.65

2.45

 

Table 2: Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration (mg/L)

Flask No.

mg O2/L after n days of exposure

7

14

21

28

Test material

1.9

1a

0.00

0.05

0.10

0.10

1b

0.20

0.15

0.20

0.30

Reference material

3.6

2a

3.80

4.55

5.10

5.30

2b

4.10

4.85

5.30

5.50

Toxicity control

Test material: 1.9, Reference material: 3.6

4a

4.30

4.75

5.10

5.20

4b

4.30

4.75

5.20

5.30

oxygen depletion: (mt0 - mtx) - (mb0 - mbx), where:

mt0 : oxygen concentration (mg/L) of test group on day 0 (1a, 2a, 4a and 1b, 2b, 4b from Table 1)

mtx: oxygen concentration (mg/L) of test group on day x (1a, 2a, 4a and 1b, 2b, 4b from Table 1)

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 1)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 1)

 

Table 3: BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration (mg/L)

Flask No.

BOD after n days of exposure

7

14

21

28

Test material

1.9

1a

0.00

0.03

0.05

0.05

1b

0.11

0.08

0.11

0.16

Reference material

3.6

2a

1.06

1.26

1.42

1.47

2b

1.14

1.35

1.47

1.53

Toxicity control

Test material: 1.9, Reference material: 3.6

4a

0.78

0.86

0.93

0.95

4b

0.78

0.86

0.95

0.96

Table 4: 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 material

1.9

1a

0.0

0.8

1.6

1.6

1b

3.2

2.4

3.2

4.9

mean

1.6

1.6

2.4

3.2

Reference material

3.6

2a

63.3

75.8

85.0

88.3

2b

68.3

80.8

88.3

91.7

mean

65.8

78.3

86.7

90.0

Toxicity control

Test material: 1.9, Reference material: 3.6

4a

31.9

35.2

37.8

38.5

4b

31.9

35.2

38.5

39.3

mean

31.9

35.2

38.2

38.9

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of this study, the test material is considered not readily biodegradable.
Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301D, EU Method C.4-E and EPA OPPTS 835.3110, under GLP conditions in a Closed Bottle Test over a period of 28 days.

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference material Sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the test material reached a mean of 3.2 % after 28 days based on the measured COD of the test material. Therefore the test material is considered not readily biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.

The reference material Sodium benzoate was sufficiently degraded to a mean of 78.3 % after 14 days, and to a mean of 90.0 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test material and the reference material Sodium benzoate, a mean of 35.2 % biodegradation was noted within 14 days and 38.9% biodegradation after 28 days of incubation.

According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 1.9 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days. The study met the validity criteria.

The test material was not considered to have significant inhibitory effects of the secondary effluent microorganisms.

Under the conditions of this study, the test material is considered not readily biodegradable.

Description of key information

Under the conditions of this study, the test material is considered not readily biodegradable.

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 material was investigated in accordance with the standardised guidelines OECD 301D, EU Method C.4-E and EPA OPPTS 835.3110, under GLP conditions in a Closed Bottle Test over a period of 28 days. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference material Sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the test material reached a mean of 3.2 % after 28 days based on the measured COD of the test material. Therefore the test material is considered not readily biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.

The reference material Sodium benzoate was sufficiently degraded to a mean of 78.3 % after 14 days, and to a mean of 90.0 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test material and the reference material Sodium benzoate, a mean of 35.2 % biodegradation was noted within 14 days and 38.9% biodegradation after 28 days of incubation.

According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 1.9 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days. The study met the validity criteria.

The test material was not considered to have significant inhibitory effects of the secondary effluent microorganisms.

Under the conditions of this study, the test material is considered not readily biodegradable.

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