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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:
02 September 2010 - 30 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study, conducted to recognised guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
The inoculum: Activated sludge, microorganisms from a domestic waste water treatment plant.

Origin: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.

Conditioning: 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 (see below) and then aerated until use. Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial conc.:
2.6 mg/L
Based on:
other: (ThODNH4): 2.34 mg O2/mg test item and on the performed 14-d preliminary test
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
RECONSTITUTED WATER

Stock solutions

In deionised water analytical grade salts were added to prepare the following stock solutions:
a) Solution: KH2PO4 2.125 g
K2HPO4 5.4375 g
Na2HPO4 x 12H2O 16.795g
NH4Cl 0.125 g
Deionised water ad 250 mL
b) Solution: MgSO4 x 7 H2O 5.625g
Deionised water ad 250 mL
c) Solution: CaCl2 x 2 H2O 9.10g
Deionised water ad 250 mL
d) Solution: FeCl3 x 6 H2O 0.25 g
Deionised water ad 1000 mL


Chemicals Used for Reconstituted Water: See table 1 below.

Ratio of ingredients

1 mL of the stock solutions a) - d) were combined and filled to a final volume of 1000 mL with deionised 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 8.4 mg/L at about 22 °C.


TEST SYSTEM

TEST UNITS

Type and Size: BOD bottles (300 ml) with special neck and glass stoppers.
Identification: Each test flask was uniquely identified with study code, test group, days of measurement and replicate number.

TEST CONDITIONS

The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ¿ 2¿C according to guideline. The test flasks were placed into an incubator and kept at 20.2-23.6¿C, in the dark. The temperature was measured on weekdays during the experiment.
The oxygen concentration of test water was 8.4 mg/L at the start of the test.
The pH value of the test water was checked prior study start. The pH of the test water was 7.39.
The test conditions were measured with suitable instruments and documented in the raw data.

PREPARATION OF THE TEST SOLUTIONS

The respective amount of Kronitex TXP was weighed in directly to reach the required test item concentration of 2.6 mg/L.
During the performance of the test the test solutions were ultrasonicated for 5 min. to ensure a good dispersion.
The chosen test item concentration was based on the theoretical oxygen demand (ThODNH4): 2.34 mg O2/mg test item and on the performed 14-d preliminary test.

The components were applied in the amounts/volumes following ratio in the test flasks:

1.) Test Item (flasks 1a and 1b)

Based on the theoretical oxygen demand (ThODNH4) of 2.34 mg O2/mg test item, 9.0 mg of Kronitex TXP was thoroughly mixed into 3.48 litres of aqueous test medium (corresponding to 2.6 mg/L test item, respectively a ThODNH4 of about 6.084 mg O2/L).

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)

Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), stock solution* corresponding to 12.096 mg of Sodium benzoate was mixed into 3.36 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L).

* The concentration of the stock solution was: 360 mg/L.


3.) Inoculum Control (flasks 3a and 3b)


Only filtered inoculum was added to 3.40 litres of aqueous test medium.

4.) Toxicity Control (flasks 4a and 4b)

9.0 mg of Kronitex TXP and reference item stock solution* (34.8 mL) were mixed into 3.48 litres of aqueous test medium corresponding to 2.6 mg/L test item (ThODNH4 of 6.084 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L).

* The concentration of the reference item stock solution was: 360 mg/L.

Microbial inoculum (0.5 ml per litre) was added to each preparation bottle.


COURSE OF THE TEST


Preparation of Test Flasks

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 standing for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stopper.


The Test Bottles

The number of test bottles was the follow:
10 bottles containing the test item and inoculum
10 bottles containing the reference item and inoculum (procedure control)
10 bottles containing only inoculum (inoculum control)
10 bottles containing the test item, reference item and inoculum (toxicity control)

MEASUREMENTS

Measurement of Oxygen

The incubation period of the closed bottle test was 28 days.
The oxygen concentrations were measured with oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.

Measurement of Temperature

Temperature was measured continuously and registered on weekdays.


CALCULATION OF RESULTS

Calculation of BOD

The BOD (mg O2 per mg test item) expected after each period was calculated.

Calculation of Biodegradation %

The percentage biodegradation of the test item and of the reference item was calculated.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
In the pretest with Kronitex TXP achieved a biodegradation of 1.6%. No further information is available.
Test performance:
See below
Key result
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
28 d
Remarks on result:
other: Not readily biodegradable
Details on results:
Under the test conditions the percentage biodegradation of Kronitex TXP reached a mean of 14.0 % after 28 days based on its ThODNH4.
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.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 40.3 % biodegradation was noted within 14 days and 40.7 % 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 2.6 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.
Key result
Parameter:
ThOD
Value:
2.34 mg O2/g test mat.
Remarks on result:
other: Based on ThOD NH4
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 71.7 % after 14 days, and to a mean of 77.5 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

RESULTS

 

 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

8.2

7.6

7.2

7.0

6.5

2.6

1b

8.3

7.7

7.2

7.2

6.4

 

mean

8.25

7.65

7.20

7.10

6.45

Reference item

 

2a

8.0

3.4

3.3

2.9

2.4

3.6

2b

8.1

3.3

3.1

2.7

2.5

 

mean

8.05

3.35

3.20

2.80

2.45

Inoculum control

3a

8.2

7.9

7.6

7.7

7.2

3b

8.1

7.9

7.6

7.5

7.2

mean

8.15

7.90

7.60

7.60

7.20

Toxicity control

Test item: 2.6
Reference item: 3.6

4a

8.1

2.7

2.5

2.4

2.0

4b

8.1

2.9

2.6

2.2

2.2

mean

8.10

2.80

2.55

2.30

2.10

 

 

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

2.6

1a

0.35

0.45

0.65

0.75

1b

0.35

0.55

0.55

0.95

Reference item

3.6

2a

4.35

4.15

4.55

4.65

2b

4.55

4.45

4.85

4.65

Toxicity control

Test item: 2.6
Reference item: 3.6

4a

5.15

5.05

5.15

5.15

4b

4.95

4.95

5.35

4.95

 

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

2.6

1a

0.13

0.17

0.25

0.29

1b

0.13

0.21

0.21

0.37

Reference item

3.6

2a

1.21

1.15

1.26

1.29

2b

1.26

1.24

1.35

1.29

Toxicity control

Test item: 2.6
Reference item: 3.6

4a

0.83

0.81

0.83

0.83

4b

0.80

0.80

0.86

0.80

 

BOD = = mg O2/mg T.i and/or

where:

T.i.                         = test item

R.i.                         = reference item

i.control                 = inoculum control

 

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

5.8

7.4

10.7

12.3

2.6

1b

5.8

9.0

9.0

15.6

 

mean

5.8

8.2

9.9

14.0

Reference item

 

2a

72.5

69.2

75.8

77.5

3.6

2b

75.8

74.2

80.8

77.5

 

mean

74.2

71.7

78.3

77.5

Toxicity control

Test item: 2.6
Reference item: 3.6

4a

41.5

40.7

41.5

41.5

4b

39.9

39.9

43.1

39.9

mean

40.7

40.3

42.3

40.7

Biodegradation % =

where:

T.i.                         = test item

R.i.                         = reference item

i.control                 = inoculum control

 

ThODNH4of test item                                = 2.34 mg O2/mg test item

ThODNH4of reference item = 1.67 mg O2/mg reference item

 The biodegradation in the toxicity control was calculated according to the following formula:

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Under the test conditions the percentage biodegradation of Kronitex TXP reached a mean of 14.0 % after 28 days based on its ThODNH4.
The test item can therefore be considered to be not ready biodegradable.
Executive summary:

The test item Kronitex TXP was investigated for its ready biodegradability 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 item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from the sewage plant for domestic sewage. The sludge was washed by centrifugation filtered through cotton wool and aerated until use. Based on the determined dry weight content, the washed sludge suspension contained 4 g dry material per litre.

Under the test conditions the percentage biodegradation of Kronitex TXP reached a mean of 14.0 % after 28 days based on its ThODNH4.

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.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 40.3 % biodegradation was noted within 14 days and 40.7 % 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 2.6 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.

Description of key information

Under the test conditions the percentage biodegradation of Kronitex TXP reached a mean of 14.0 % after 28 days based on its ThODNH4.

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 test item Kronitex TXP was investigated for its ready biodegradability 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 item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from the sewage plant for domestic sewage. The sludge was washed by centrifugation filtered through cotton wool and aerated until use. Based on the determined dry weight content, the washed sludge suspension contained 4 g dry material per litre.

Under the test conditions the percentage biodegradation of Kronitex TXP reached a mean of 14.0 % after 28 days based on its ThODNH4.

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.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 40.3 % biodegradation was noted within 14 days and 40.7 % 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 2.6 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.