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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:
2006-11-24 to 2007-05-25
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
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
1992
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): STP for domestic sewage in Veszprém, Hungary
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 4 g dry material per litre were mixed with test water and then aerated until use. Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial conc.:
2.5 mg/L
Based on:
test mat.
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
A first test was conducted with the concentration of 2.5mg/L. Due to the observed degradation < 25% (23.3%) the test item was assumed to be inhibitory on the activated sludge microorganisms. The test was repeated with a lower concentration of 2.0 mg/L.

TEST CONDITIONS
- Composition of medium: grade salts were added to deionised water to give stock solutions. 1 mL of stock solutions were combined and filled to final volume of 1000 mL with deionised water. The test medium was aerated for 20 minutes and allowed to stand for about 20 hrs at the test temperature. Dissolved oxygen concentration was about 9.3 mg/L at about 20°C (in both experiments).
- Test temperature: 19.1-21.0°C; 19.4-21.0°C (in the additional experiment)
- pH: 7.49 (measured at the start of the test); 7.48 (measured at the start of the additional test)
- Aeration of dilution water: yes (see above "composition of medium")

TEST SYSTEM
- Culturing apparatus: BOD 300 mL bottles
- Number of culture flasks/concentration: Preparation of Test Flasks: Each 16 (+2 reserve) (10 (+2 reserve) in the additional test) bottles containing: a) the test item and inoculum, b) the reference item and inoculum (positive control), c) only inoculum (inoculum control) and d) test item, reference item and inoculum (toxicity control).
Preparation of the Test Solutions: Test Item (flasks 1a and 1b): Based on the theoretical oxygen demand (ThODNO3) of 2.41 mg O2/mg test item (calculated according to equation given in the guidelines), stock solution corresponding to 17.3 mg of the test item was thoroughly mixed into 6.92 litres of aqueous test medium (corresponding to 2.5 mg/L test item, respectively a ThODNO3 of about 6.03 mg O2/L). In the additional test 13.84 mg of the test item was thoroughly mixed into 6.92 litres of aqueous test medium (corresponding to 2.0 mg/L test item and a ThODNO3 of about 4.82 mg O2/L). Procedure Control: Sodium acetate (flasks 2a and 2b): Based on the theoretical oxygen demand (ThODNH4) of Sodium acetate (0.78 mg O2 per mg) (details on calculation are given in the guidelines), stock solution corresponding to 52.896 mg of Sodium acetate was mixed into 6.96 litres of aqueous test medium (corresponding to 7.6 mg/L reference item and a ThODNH4 of about 5.93 mg O2/L). Inoculum Control (flasks 3a and 3b): Only filtered inoculum was added to 6.80 litres of aqueous test medium. Toxicity Control (flasks 4a and 4b): Test and reference item stock solutions (17.3 mg of the test item and 52.592 mg of Sodium acetate) were mixed into 6.92 litres of aqueous test medium corresponding to 2.5 mg/L test item (ThODNO3 of 6.03 mg O2/L) and 7.6 mg/L reference item (ThODNH4 of 5.93 mg O2/L). In the additional test 13.84 mg of the test item and 52.592 mg of Sodium acetate) was mixed into 6.92 litres of aqueous test medium corresponding to 2.0 mg/L test item (ThODNO3 of 4.82 mg O2/L) and 7.6 mg/L reference item (ThODNH4 of 5.93 mg O2/L). General: In the first assay a microbial inoculum (0.05 ml per litre) was added to each preparation bottle. The volume of microbial inoculum added to each preparation bottle was 0.1 ml per litre in the additional experiment.
- Method used to create aerobic conditions: inoculum and medium solutions were sufficiently aerated prior to the start of the test.
- Measuring equipment: The oxygen concentrations will be measured with oxygen meter with a stirring O2 electrode.
- Test performed in closed vessels: yes

SAMPLING
- Sampling method/frequency: Due to the nitrogen content of the test item, samples for nitrate and nitrite analysis were taken from all vessels (of test item, inoculum control and toxicity control group) and the total oxidised nitrogen (nitrate and nitrite) concentrations were determined after each oxygen measurement.
- Sample storage before analysis: Samples were taken after the oxygen measurements from each test vessel, and stored in a refrigerator immediately after sampling, until determination of the total oxidised Nitrogen concentrations.
- Other: Nitrite and nitrate concentrations were measured (representing oxidised nitrogen within the test solutions) via HPLC-UV analysis. Samples were directly injected.
- Other: Oxygen: measurements were performed in duplicate on days 0, 2, 5, 7, 12, 14, 21 and 28; in the additional test the oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28. Temperature: were measured each day during the test period.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no data
- Toxicity control: yes

Reference substance:
acetic acid, sodium salt
Preliminary study:
The ready biodegradability of the test item was examined in a Closed Bottle Test over a period of 28 days withe a concentration of 2.5 mg test item/L. The validity criteria of the study were fulfilled in this experiment, however in the toxicity control containing both, the test item and the reference item Sodium acetate, a mean of 23.3 % biodegradation was noted within 14 days. According to the test guidelines the test item was assumed to be inhibitory on the activated sludge microorganisms because
degradation was < 25 % within 14 days. Due to the inhibition observed the test series was repeated. An additional test was performed using lower concentration of test item and higher concentration of inoculum, under otherwise the same conditions as the first test.
Key result
Parameter:
% degradation (O2 consumption)
Value:
24.9
Sampling time:
28 d
Remarks on result:
other: Additional test
Details on results:
The residual oxygen concentration in the test flask did not drop below 0.5 mg O2/L at any time.

The BOD values were not corrected for nitrification, because the quantity of the total oxidised Nitrogen (nitrite and nitrate) was below the LOQ or was about 0 after the correction with control values and the occurrence of total oxidised Nitrogen (nitrite and nitrate) in each group (based on the analytical measurements) did not appear at the biological results (dissolved oxygen depletions).
Results with reference substance:
Biodegradation of Reference Item in the Additional Experiment:
The reference item Sodium acetate was sufficiently degraded to a mean of 69.1 % after 14 days, and to a mean of 103.7 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the activated sludge inoculum used in the performed additional test.

Biodegradation of Toxicity Control in the Additional Experiment:
In the toxicity control containing both the test item and the reference item Sodium acetate, a mean of 25.8 % biodegradation was noted within 14 days and 36.3 % biodegradation after 28 days of incubation of additional test.
According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25 % within 14 days.
Thus, the test item can be assumed to not inhibit the activated sludge microorganisms at the examined concentration level.

Table 1: Percentage biodegradation at different time intervals during the exposure period of 28 days (First test)

Treatment

Concentration [mg/L]

Flask No.

Percent of biodegradation after n days of exposure

2

5

7

12

14

21

28

Test item

2.5

1a

1.7

2.5

0.8

0.0

-1.7

-1.7

-0.8

1b

-1.7

-0.8

0.8

0.0

-3.3

-5.0

-4.2

Mean

0.0

0.8

0.8

0.0

-2.5

-3.3

-2.5

Reference item

7.6

2a

52.3

70.0

70.0

72.5

70.8

75.9

81.8

2b

48.9

68.3

71.6

72.5

70.8

75.9

80.1

Mean

50.6

69.1

70.8

72.5

70.8

75.9

80.9

Toxicity control

Test item: 2.5 Reference item: 7.6

4a

19.2

23.9

23.3

23.6

23.0

25.5

25.1

4b

19.9

25.1

24.5

24.2

23.6

25.5

25.8

Mean

19.6

24.5

23.9

23.9

23.3

25.5

25.5

Table 2: Percentage biodegradation at different time intervals during the exposure period of 28 days of the additional test (Additional test):

Treatment

Concentration [mg/L]

Flask No.

Percent of biodegradation after n days of exposure

7

14

21

28

Test item

2.0

1a

4.2

-1.0

23.9

21.8

1b

8.3

3.1

34.3

28.0

Mean

6.2

1.0

29.1

24.9

Reference item

7.6

2a

72.5

68.3

107.1

105.4

2b

72.5

70.0

102.0

102.0

Mean

72.5

69.1

104.5

103.7

Toxicity control

Test item: 2.0 Reference item: 7.6

4a

25.5

26.5

36.3

36.9

4b

23.5

25.2

35.6

35.6

Mean

24.5

25.8

35.9

36.3

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item is not readily biodegradable based on 24.9 % biodegradation in 28 days.

Description of key information

The test item is not readily biodegradable based on a reliable biodegradation study according to OECD 301 D with  24.9 % biodegradation in 28 days.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A biodegradation value of 24.9 % in 28 days was determined in a reliable study conducted according to OECD 301 D (Closed Bottle Test) and in compliance with GLP. In this test the ready biodegradability of the test item was examined. In the toxicity control containing both the test item at 2.5 mg/l and reference item, an inhibitory effect on the activated sludge microorganisms was observed. Therefore an additional study was conducted using a lower concentration of test item (2 mg/L) and a higher concentration of inoculum. As the percentage biodegradation of the test item reached a mean of 24.9% after 28 days based on ThOD (NO3), the test item can be considered to be not ready biodegradable. No inherent biodegradation test is available and no evidence of inherent biodegradation can be determined based on the result obtained from the ready biodegradability test, thus a worst case approach is considered and the key value no biodegradation observed is used for chemical safety assessment.