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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
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Remarks:
The study was conducted according to the test guidelines in effect at the time of study conduct.
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Remarks:
The study was conducted according to the test guidelines in effect at the time of study conduct.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
A sample of activated sludge was obtained from Worlingworth sewage treatment works (Suffolk, UK), which treats predominantly domestic waste. At the time of collection, the sludge was sieved (1 mm2) then transported to the laboratory and left to 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. The concentration of suspended solids in a blended and homogenous sample was determined before the start of the test. Aliquots (10 mL) of the sludge were filtered through dried and preweighed Whatman GF/C filters in triplicate, which were then dried again at approximately 105°C for one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the sludge was then determined and the volume required to give a solids level of 30 mg/L in test cultures was calculated.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
other: ThOD
Details on study design:
One vessel was used for the reference and the inhibition assay. Two vessels were used for the blank control and the test substance. The test substance (309 mg) was added to ultra high purity water in a volumetric flask (100 mL) and made up to volume with mixing to give a stock solution (3.09 mg/mL). Aliquots (10 mL) of this stock solution were added to their respective test vessels to give, after making up to full test volume with inoculated MSM, a nominal oxygen demand of 25 mg ThOD/500 mL equivalent to 50 mg ThOD/L. Sodium benzoate (20 mL) was added as an aqueous stock solution (0.750 g/L) in MSM to the reference and inhibition assay cultures to give a final nominal concentration of 25 mg ThOD/500 mL equivalent to 50 mg ThOD /L. The pH of the cultures was measured and no adjustment was necessary. The CO2 absorber was replaced in each cell (to ensure the maximum adsorption capacity in the test) and the cultures were sealed and returned to the water bath to equilibrate. The cells were connected to the computer-controlled system and the test was initiated. A record of the cumulative oxygen demand made by each cell was printed at hourly intervals. The correct operation of the magnetic stirrers and the temperature of the water bath were recorded at appropriate intervals, typically daily, during the test. The nominal temperature range for the test was 22±2ºC. On Day 28, the test was halted and the pH of each mixture was measured.

Validity criteria:
Biodegradation of the reference substance alone should be ≥ 60% by Day 14.
Oxygen consumption by the controls should be, typically, 20 to 30 mg O2/L but should not exceed 60 mg O2/L.
Biodegradation of the reference substance in the presence of the test substance (inhibition assay) should not be < 25% on Day 14.
The difference between extremes of replicate values for the removal of the test substance at the plateau, at the end of the test or at the end of the 10-day window, as appropriate, should be ≤ 20%. This only applies where the test substance is considered to be readily biodegradable.
Reference substance:
benzoic acid, sodium salt
Test performance:
The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 15.45 mg O2/500 mL or 62% of the ThOD (25 mg O2/500 mL) after 4 days of incubation. In the presence of the test substance, degradation of sodium benzoate had achieved 61% by Day 5. Cumulative levels of oxygen consumption by the controls after 28 days (15.70 and 14.39 mg O2/500 mL, equivalent to 31.40 and 28.78 mg O2/L) were considered to be acceptable for this assay system. These results confirm that the test substance was not inhibitory to the activity of the microbial inoculum and that the test was valid.
Key result
Parameter:
other: % biodegradation
Value:
0
Sampling time:
28 d
Details on results:
There was no blank-corrected oxygen consumption in mixtures containing the test substance during the period of the test (28 days). Substances are considered to be readily biodegradable in this type of test if oxygen consumption is equal to or greater than 60% of the ThOD of the test mixtures within ten days of the consumption achieving 10%. Therefore, the test substance was considered not to be readily biodegradable under the conditions of this test.
Results with reference substance:
In the presence of the test substance, degradation of sodium benzoate had achieved 61% by Day 5.

Respirometry test – biodegradation of the test and reference substances

Day

Test substance (50 mg O2/L)

Sodium benzoate

(50 mg ThOD/L)

Sodium benzoate

(50 mg ThOD/L)

in the inhibition mixture

Replicate 1

Replicate 2

Mean

0

0

0

0

0

0

1

1

0

0

8

20

2

0

0

0

52

53

3

0

0

0

57

55

4

0

0

0

62

58

5

0

0

0

67

61

6

0

0

0

71

63

7

0

0

0

75

64

8

0

0

0

78

65

9

0

0

0

80

66

10

0

0

0

82

66

11

0

0

0

85

66

12

0

0

0

87

66

13

0

0

0

88

66

14

0

0

0

90

66

15

0

0

0

91

66

16

0

0

0

92

66

17

0

0

0

93

66

18

0

0

0

94

67

19

0

0

0

95

67

20

0

0

0

95

67

21

0

0

0

96

67

22

0

0

0

97

67

23

0

0

0

98

68

24

0

0

0

98

68

25

0

0

0

99

68

26

0

0

0

100

68

27

0

0

0

100

68

28

0

0

0

100

68

The data presented were calculated using unrounded values stored in the computer database. Minor numerical differences may be observed if rounded values are used to calculate the data. This minor discrepancy is not considered to be significant.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance was not readily biodegradable.
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The ready biodegradability of the test substance was assessed in accordance with Directive (EC) 440/2008, C.4-D, ‘Determination of Ready Biodegradability, Manometric Respirometry’ and OECD Test Guideline 301F ‘Ready Biodegradability, Manometric Respirometry Test’, adopted 17 July 1992. The test substance was added to two bottles containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 61.7 mg /L, equivalent to 50 mg O2/L. Two control cultures contained inoculated mineral salts medium alone. Two cultures contained inoculated mineral salts medium plus the reference substance sodium benzoate (50 mg O2/L) of which one also contained the test substance (50 mg O2/L) in order to assess the potential inhibitory effects of the test substance on the microbial inoculum. The test system comprised of an automated system for oxygen (O2) generation and the cultures were stirred and held in a thermostatically-controlled water bath.

The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 15.45 mg O2/500 mL or 62% of the ThOD (25 mg O2/500 mL) after 4 days of incubation. In the presence of the test substance, degradation of sodium benzoate had achieved 60% by Day 5. Cumulative levels of oxygen consumption by the controls after 28 days (15.70 and 14.39 mg O2/500 mL, equivalent to 31.40 and 28.78 mg O2/L) were considered to be acceptable for this assay system. These results confirm that the test substance was not inhibitory to the activity of the microbial inoculum and that the test was valid. There was no blank-corrected oxygen consumption in mixtures containing the test substance during the period of the test (28 days). Substances are considered to be readily biodegradable in this type of test if oxygen consumption is equal to or greater than 60% of the ThOD of the test mixtures within ten days of the consumption achieving 10%. Therefore, the test substance was considered not to be readily biodegradable under the conditions of this test.

Description of key information

Not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

In an OECD Guideline 301F study, the test substance was determined to be not readily biodegradable (0% biodegradation after 28 days).