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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
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)
Deviations:
yes
Remarks:
(on four measurements the temperatures recorded for the water bath were below the recommended minimum temperature. As the validity criteria were met, this deviation was not considered to have affected the integrity of the study.)
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
yes
Remarks:
(on four measurements the temperatures recorded for the water bath were below the recommended minimum temperature. As the validity criteria were met, this deviation was not considered to have affected the integrity of the study.)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Worlingworth sewage treatment works (Suffolk, UK)
- Method of cultivation / Preparation of inoculum for exposure: At the time of collection, the sludge was sieved (1 mm²) then transported to the laboratory and left 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.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
ThOD/L
Initial conc.:
18 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral salts medium (MSM) according to guideline, prepared using tap-water that had been softened and treated by reverse osmosis and then purified; nominal resistivity ≥ 18 MegOhm.cm. The pH was adjusted to 7.56 with 5M HCl. This water complies with the relevant standards (British Standard (BS) 3978, 1987; American Society for Testing and Materials (ASTM) D 1193-06) for classification as Grade 1 or Type 1 water for laboratory use.
- Test temperature: 21.3 to 21.9°C. On Days 6, 15, 20, and 28 the minimum temperatures recorded for the water bath were below this range (18.2, 12.2, 18.2 and 18.0°C respectively). These low temperature values were considered anomalous as the ambient room temperature over the course of the study was considered acceptable. The reasons for these low recorded values are unknown. As the validity criteria for the study were met, this deviation from protocol is not considered to have affected the integrity of the study.
- pH: 7.5 at the start of the test and 7.5 to 8.1 at the end
- pH adjusted: not necessary
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes (amber glass bottles)
- Other: The inoculum was added to the culture bottles 1 day before test initiation to allow a period of ageing.

TEST SYSTEM
- Culturing apparatus: amber glass culture bottles in a thermostatically controlled water bath
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: internal oxygen generating process
- Measuring equipment: Co-ordinated Environmental Services (CES) Ltd automated respirometer with associated software to monitor the cumulative amount of oxygen consumed

SAMPLING
- Sampling frequency: continuous monitoring of the cumulative amount of oxygen consumed; the cumulative oxygen demand made by each cell was printed at, typically, hourly intervals

CONTROL AND BLANK SYSTEM
- Inoculum blank (2 flasks): inoculated mineral salts medium (MSM)
- Reference (1 flask): inoculated MSM + sodium benzoate (50 mg O₂/L)
- Toxicity control (1 flask): inoculated MSM + test material (50 mg O₂/L) + sodium benzoate (50 mg O₂/L)
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
6
Sampling time:
28 d
Details on results:
Due to the complex nature, and owing the low nitrogen content of the test material, there was considered to be no indication that any nitrification observed would be significant.
In the presence of test material the degradation of sodium benzoate achieved 62% after 3 days indicating that the test substance was not inhibitory to the microbial inoculum (Table 1).
Results with reference substance:
The reference substance sodium benzoate had achieved 64% of the ThOD after 3 days of incubation and 101% by Day 28 (Table 1).

Table 1: Biodegradation of the test and reference substances

 

% Biodegradation

Day

Test material

(50 mgO2/L)

Sodium Benzoate (50 mgO2/L)

Sodium Benzoate

(50 mgO2/L)

in the inhibition mixture

Test 1

Test 2

Mean

1

1

1

1

38

46

2

2

1

1

57

55

3

2

1

2

64

62

4

1

2

1

70

-

5

1

2

2

74

-

6

2

2

2

78

-

7

2

2

2

80

-

8

3

2

3

82

-

9

3

2

3

84

-

10

3

3

3

85

-

11

3

3

3

87

-

12

4

3

3

88

-

13

4

3

3

89

-

14

4

3

3

90

-

15

4

3

3

91

-

16

4

3

4

92

-

17

4

3

4

93

-

18

5

4

4

94

-

19

5

4

4

95

-

20

5

4

5

96

-

21

5

4

5

96

-

22

5

5

5

97

-

23

5

5

5

98

-

24

5

5

5

99

-

25

5

5

5

99

-

26

5

5

5

100

-

27

6

5

6

101*

-

28

6

6

6

101*

-

 *: Value slightly exceeds 100% of the available concentration. However this was not considered significant as this is within the level of experimental error for this type of study.

Validity criteria fulfilled:
yes
Remarks:
the removal of the test substance in replicate values differed less than 20%, the reference compound reached the pass level by day 3, and the cumulative amount of oxygen consumed by the control mixtures was 35-36 mg O₂/L
Interpretation of results:
under test conditions no biodegradation observed

Description of key information

Biodegradation in water: screening tests: biodegradation <10% (ThOD) in 28 days (OECD 301F, EU C4-D)

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable

Additional information

.