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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:
29 July 2010 to 26 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP study conducted to current accepted guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.

- Pretreatment: The activated sludge used for this study was washed by centrifugation (for 10 min.) and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged (for 10 min.). 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 4 g dry material per litre were mixed with test water and then aerated until use.

- Concentration of sludge: the washed sludge suspension contained 4 g dry material per litre. Microbial inoculum at 0.5 ml per litre was added to each preparation bottle.

- Type and size of filter used, if any: Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial conc.:
7.5 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 mL of the stock solutions a) - d) were combined and filled to a final volume of 1000 mL with deionised water. Table 1, under section 'Any other information on results incl. tables', lists the composition of the stock solution for preparation of the reconstituted medium.
- Test temperature: Flasks were placed in an incubator at 21.6-23 °C
- pH: The pH of the test water was 7.21.
- Aeration of dilution 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 about 8.5 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BOD flasks
- Number of culture flasks/concentration: 10 per preparation
- Method used to create aerobic conditions: The reconstituted medium was aerated for 20 minutes (then allowed to stand for 20 hours) prior to use. The test inoculum, after pre-treatment was aerated until use.
- Measuring equipment: The COD (chemical oxygen demand) of the test item were determined in using Lovibond® COD Measuring System. (The oxygen concentrations were measured with oxygen meter with a stirring O2 electrode)
- Test performed in open system: No, the test bottles were sealed tightly with a glass stopper

MEASUREMENTS
- Measurement frequency: Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28. Temperature was measured continuously and registered on weekdays.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, 10 bottles containing only inoculum (inoculum control)
- Procedure control: Yes, 10 bottles containing the reference item and inoculum (procedure control)
- Toxicity control: Yes, 10 bottles containing the test item, reference item and inoculum (toxicity control)

STATISTICAL METHODS:
Calculation of BOD
The BOD (mg O2 per mg test item) expected after each period was calculated as follows:

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

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

Calculation of Biodegradation %

The percentage biodegradation of the test item and of the reference item was calculated as follows:

(BOD (mg O2/mg T.i. or R.i.)/ COD (mg O2/mg T.i.) or ThODNH4 (mg O2/mg R.i.)) x 100

where:
T.i. = test item
R.i. = reference item,
i.control = inoculum control
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The chosen test item concentration was based on the measured chemical oxygen demand (COD): 0.8 ±0.04 mg O2/mg test item and on the performed 14-d preliminary test. No further details are reported.
Test performance:
No data available
Key result
Parameter:
% degradation (O2 consumption)
Value:
3.3
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of Great Lakes DBS reached a mean of 3.3 % after 28 days based on the measured COD of the test item.
The test item can therefore be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.

COD of test item = 0.8 ± 0.04 mg O2/mg test item
ThODNH4 of reference item = 1.67 mg O2/mg reference item
Parameter:
COD
Value:
0.8 other: mg O2/mg test material
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 75.0 % after 14 days, and to a mean of 79.2 % 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 Sodium benzoate, a mean of 28.9 % biodegradation was noted within 14 days and 29.6 % biodegradation after 28 days of incubation.

Table 2.  Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

mg O2/L after n days of exposure

[mg/L]

No.

0

7

14

21

28

Test item

 

1a

8.3

7.7

7.4

7.2

7.1

7.5

1b

8.4

7.9

7.4

7.1

7.0

 

mean

8.35

7.80

7.40

7.15

7.05

Reference item

 

2a

8.4

3.4

3.1

2.8

2.5

3.6

2b

8.4

3.4

3.1

2.7

2.6

 

mean

8.40

3.40

3.10

2.75

2.55

Inoculum control

3a

8.4

8.1

7.6

7.6

7.4

3b

8.4

8.1

7.6

7.5

7.2

mean

8.40

8.10

7.60

7.55

7.30

Toxicity control

Test item: 7.5
Reference item: 3.6

4a

8.3

3.8

3.6

3.5

3.1

4b

8.3

3.8

3.5

3.5

3.2

mean

8.30

3.80

3.55

3.50

3.15

 

 

Table 3.  Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

mg O2/L after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

7.5

1a

0.30

0.10

0.25

0.10

1b

0.20

0.20

0.45

0.30

Reference item

3.6

2a

4.70

4.50

4.75

4.80

2b

4.70

4.50

4.85

4.70

Toxicity control

Test item: 7.5
Reference item: 3.6

4a

4.20

3.90

3.95

4.10

4b

4.20

4.00

3.95

4.00

 

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

 

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

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

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 2)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 2)

 

Table 4.  BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

BOD after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

7.5

1a

0.04

0.01

0.03

0.01

1b

0.03

0.03

0.06

0.04

Reference item

3.6

2a

1.31

1.25

1.32

1.33

2b

1.31

1.25

1.35

1.31

Toxicity control

Test item: 7.5
Reference item: 3.6

4a

0.38

0.35

0.36

0.37

4b

0.38

0.36

0.36

0.36

 

Table 5.  Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

Percent of biodegradation after n days of exposure

 

[mg/L]

No.

7

14

21

28

Test item

 

1a

5.0

1.7

4.2

1.7

7.5

1b

3.3

3.3

7.5

5.0

 

mean

4.2

2.5

5.8

3.3

Reference item

 

2a

78.3

75.0

79.2

80.0

3.6

2b

78.3

75.0

80.8

78.3

 

mean

78.3

75.0

80.0

79.2

Toxicity control

Test item: 7.5
Reference item: 3.6

4a

30.7

28.5

28.9

29.9

4b

30.7

29.2

28.9

29.2

mean

30.7

28.9

28.9

29.6

Validity criteria fulfilled:
yes
Remarks:
The study fulfilled the validity
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Under the test conditions the percentage biodegradation of Great Lakes DBS reached a mean of 3.3 % after 28 days based on the measured COD of the test item. The test item can therefore be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.

The reference item Sodium benzoate was sufficiently degraded to a mean of 75.0 % after 14 days, and to a mean of 79.2 % 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 Sodium benzoate, a mean of 28.9 % biodegradation was noted within 14 days and 29.6 % biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 7.5 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.
Executive summary:

In a GLP compliant study performed to OECD 301D, the biodegradation of the test material was studied in activated sludge (domestic). Under the test conditions the percentage biodegradation of Great Lakes DBS reached a mean of 3.3 % after 28 days based on the measured COD of the test item. The test item can therefore be considered to be not ready biodegradable.

Description of key information

In a GLP compliant study performed to OECD 301D, the biodegradation of the test material was studied in activated sludge (domestic). Under the test conditions the percentage biodegradation of Great Lakes DBS reached a mean of 3.3 % after 28 days based on the measured COD of the test item. The test item can therefore be considered to be not ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The key study, Sipos K (2010), was provided as a stand-alone study to fulfil this data requirement. The study was performed in compliance with GLP and to a harmonised guideline (OECD 301D). The study was accordingly assigned a reliability score of 1 and considered reliable and adequate for assessment.