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Environmental fate & pathways

Biodegradation in water: screening tests

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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:
yes
Remarks:
(the nominal ThOD based test material concentration was 28 mg O2/L instead of > 50 mg O2/L as recommended by the testing guideline. This deviation did not adversely impact the results of this study.)
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
yes
Remarks:
(the nominal ThOD based test material concentration was 28 mg O2/L instead of > 50 mg O2/L as recommended by the testing guideline. This deviation did not adversely impact the results of this study.)
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: 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.:
28 mg/L
Based on:
ThOD/L
Initial conc.:
9.4 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 wasadjusted to 7.4 ± 0.2 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: 22 to 24°C.
- pH: 7.3-7.5 at the start of the test and 7.4-7.5 at the end
- pH adjusted: not necessary
- Suspended solids concentration: 30 mg/L
- Continuous darkness: not reported; amber glass bottles were used
- 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 temperature 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

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)
- Nitrification test (1 flask): inoculated MSM + test material (50 mg O₂/L) + Allylthiourea (ATU, 11.6 mg/L)
- Nitrification control (1 flask): inoculated MSM + ATU (11.6 mg/L)
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
87
Sampling time:
28 d
Remarks on result:
other: based on ThOD
Details on results:
Mean oxygen consumption in biotic mixtures containing the test material was equivalent to 11% of the ThOD value after approximately 1 day, 67% after 11days and 87% at the end of the test (Day 28). Thus, the test material was ready biodegradable (Table 1).
In the nitrification controls the degradation of the test material was similar to that in uninhibited cultures (Table 1)
In the presence of test material the degradation of sodium benzoate achieved 65% after 3 days indicating that the test substance was not inhibitory to the microbial inoculum.
Results with reference substance:
The reference substance sodium benzoate had achieved 61% of the ThOD after 2 days of incubation and 107% by Day 28 (Table 1).

Table 1: Biodegradation of the test and reference substances

 

% Biodegradation

Day

Test material

Test material

in the presence of ATU

Sodium Benzoate

Sodium Benzoate

in the inhibition mixture

Test 1

Test 2

Mean

1

17

6

11

0

2

4

2

33

24

29

4

61

59

3

40

31

36

24

63

65

4

49

40

44

35

67

-

5

56

46

51

40

70

-

6

60

49

55

44

73

-

7

63

52

57

47

77

-

8

66

54

60

50

81

-

9

69

56

63

52

86

-

10

72

58

65

55

90

-

11

75

59

67

57

93

-

12

77

61

69

59

96

-

13

79

62

70

61

99

-

14

81

64

72

64

101

-

15

82

65

73

65

103

-

16

84

66

75

67

104

-

17

86

67

76

69

104

-

18

87

67

77

70

104

-

19

89

68

79

71

105

-

20

91

69

80

73

105

-

21

93

69

81

74

105

-

22

94

70

82

76

105

-

23

96

71

83

77

106

-

24

97

71

84

79

106

-

25

99

72

85

81

107

-

26

100

72

86

83

107

-

27

100

72

86

84

107

-

28

102

73

87

87

107

-

 

Validity criteria fulfilled:
yes
Remarks:
rate of degradation of sodium benzoate was 61% of its ThOD after 2 days; cumulative amount of oxygen consumed by the control mixtures was 34 and 36 mg O₂/L; difference between replicates was <20% at the end of the 10-d window
Interpretation of results:
other: readily biodegradable. See Appendix 1 with additional statements.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to international guideline(s), conducted under Quality Assurance Programme; unsuitable test system; however enough information to support recent results
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
GLP compliance:
yes
Remarks:
Quality Assurance Programme/GLP standards not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: secondary effluent, domestic
Details on inoculum:
The inoculum was derived from the secondary effluent of a treatment plant receiving predominantly domestic sewage. Inoculum was collected and filtered through a folded filter. The sample was aerated for 6 days at room temperature (pre-conditioning), filtered again and used for test preparation.
Duration of test (contact time):
28 d
Initial conc.:
32 mg/L
Based on:
DOC
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium as in test guideline (containing yeast extract as source for vitamines)
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 22 °C ± 2 °C
- pH: 7.4 ± 0.1
- pH adjusted: no
- Suspended solids concentration: not determined
- Continuous darkness: no (diffuse light)

PREPARATION OF TEST MATERIAL SOLUTION
Approximately 2 g of test material was each weighed into two 1L glass-bottles. The bottles were filled up to 1 L with mineral medium. The flasks were shaken overnight on a laboratory shaker. Additional filtration through a folded filter resulted in a clear solution. The DOC of the saturated solutions was determined to 71 mg DOC/L (bottle 1) and 23 mg DOC/L (bottle 2). The pH value of the test substance solutions was determined to 7.4 ± 0.1. The prepared saturated solution of bottle 1 was diluted with mineral medium (1:2), the saturated solution of bottle 2 was used for the test without dilution. The samples wereas inoculated by addition of 0.5 mL of inoculum.

TEST SYSTEM
- Culturing apparatus: 1 L glass-bottles covered with aluminium foil to allow free exchange of air
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: samples were incubated on a laboratory shaker
- Measuring equipment: DOC analyser
- Test performed in open system: yes

SAMPLING
- Sampling frequency: Day 0 (immediately after preparation), 1, 5, 9, 16, 26, and 28
- Sampling method: A volume of about 10 ml of each test suspension was taken at sampling. Before sampling any evaporation losses from the flasks were made good by adding water in the required amount if necessary. The culture medium was thoroughly shaken before withdrawing a sample and it was ensured that no material was adhering to the walls of the vessels. The samples were membrane filtered (0.45 µm) immediately after they had been taken.
- Sample storage before analysis: filtered samples were stored at -18 C until sample analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Positive control: sodium acetate (2x); sodium benzoate (2x)
Reference substance:
benzoic acid, sodium salt
Remarks:
42.8 mg/l
Reference substance:
acetic acid, sodium salt
Remarks:
141.7 mg/l
Key result
Parameter:
% degradation (DOC removal)
Value:
84
St. dev.:
10
Sampling time:
28 d
Details on results:
Mean DOC decrease was approx. 20% on Days 1 and 5 and reached a plateau of approx. 80% on Day 16 (Table 1). The criterion for ready biodegradability was fulfilled in Flask 2 and for the mean of the two samples. However, since the test material is an UVCB the 10-day window condition (i.e. at least 70% degradation within 10 days after 10% degradation have been reached) does not apply (Commission Regulation (EU) No 286/2011).
Results with reference substance:
Both reference samples, sodium benzoate and sodium acetate, reached a degradation level of more than 70% within 10 days after the substance degradation had reached a l0% threshold degradation level.

Table 1: DOC values measured in test material flasks

 

Day

DOC¹

Degradation in %

Blank²

Flask 1

Flask 2

Flask 1

Flask 2

Mean

0

3.95

31.65

20.45

0.0

0.0

0.0

1

4.05

24.35

16.35

23.1

20.0

21.9

5

4.8

27

14.1

14.7

31.1

21.1

9

6.3

11.7

4.8

63.0

76.5

68.3

16

6.05

9.95

0.25

68.6

98.8

83.7

26

4.8

4.6

0.9

85.5

95.6

90.5

28

5.5

8.3

1.2

73.8

94.1

84.0

 

¹: Flasks 1 and 2 corrected for blank

²: Mean of two replicates

Validity criteria fulfilled:
no
Remarks:
difference of extremes of replicate values of the removal of the test substance was >20%
Interpretation of results:
other: readily biodegradable. For UVCB-substances the 10-day window condition does not apply (Commission Regulation (EU) No 286/2011).

Description of key information

Biodegradation in water: screening tests: biodegradation 87% (ThOD) and 84% (DOC) in 28 days (OECD 301F, EU C.4-D and OECD 301E, EU C.4-B)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information