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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
Study period:
11 Oct - 09 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sewage sludge micro-organisms from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK; sampled on 10 Oct 2016
- Storage conditions: Sludge was used on the day of collection.
- Storage length: No storage
- Preparation of inoculum for exposure: Sludge sample was washed twice by settlement and re-suspension in mineral medium; washed sample was continuously aerated in the laboratory at a about 21 ºC.
- Pretreatment: The activated sludge was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC).
- Concentration of sludge: 30 mg suspended solids (ss)/L
- Water filtered: yes (for determination of suspended solids)
- Type and size of filter used, if any: GF/A filter paper using a Buchner funnel
Duration of test (contact time):
29 d
Initial conc.:
22.6 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to the OECD 301
- Test temperature: 20 and 25 °C
- Aeration of dilution water: 24 h prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 34.6 mL of inoculum and aerated overnight.
- pH: 7.5 - 7.7
- pH adjusted: yes
- Suspended solids concentration: The suspended solids concentration was equal to 2.6 g/L prior to use; test vessels were inoculated with the inoculum at a final concentration of 30 mg suspended solids (ss)/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: sealed 5 L absorber vessels
- Number of culture flasks/concentration: control, reference item test item: 2 vessels and toxicity control: 1 vessel
- Method used to create aerobic conditions: CO2-free air bubbled at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer
- Measuring equipment: IC/TC ratio was measured using a Shimadzu TOC-VCPH TOC Analyzer
- Details of trap for CO2 and volatile organics if used: CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules. The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29
- Sampling method: IC/TC ratio of the test substance (only Day 0): Samples (30 mL) were filtered through 0.45 μm Gelman AcroCap filters prior to DOC analysis. The samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer, 50 µL injected
- Sample storage before analysis: samples were analyzed for IC immediately; remainder of all samples with the exception of the Day 0 samples were frozen for further analysis if required.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes

STATISTICAL METHODS: no statistical method was used
Reference substance:
benzoic acid, sodium salt
Remarks:
17.1 mg/L equivalent to 10 mg carbon/L
Parameter:
% degradation (CO2 evolution)
Value:
49
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
3
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
28
Sampling time:
6 d
Details on results:
Biodegradation
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the test guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item. The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels. This decrease was considered to be due to sampling/analytical variation. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
Results in detail are summarized in tables 1 to 3 within section "Any other information on results incl. tables"
Results with reference substance:
Sodium benzoate attained 83% biodegradation after 14 days and 84% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria:

The total CO2 evolution in the inoculum control vessels on Day 28 was 43.09 mg/L. Although the CO2 evolution was in excess of 40 mg/L at the end of the test, this was considered not to affect the integrity of the study given that the upper level of 70 mg CO2/L given in the OECD Test Guidelines was not exceeded, and that all other validation criteria were satisfied. The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines. The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

Table 1: Inorganic Carbon Values on Each Analysis Occasion

Day

Inorganic Carbon (mg IC)

 

Inoculum Control

Procedure Control

Test Item

Toxicity Control

 

R1

R2

R1

R2

R1

R2

R1

 

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

0

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.75

1.87

1.75

1.87

2

5.80

-

6.15

-

21.92

-

21.34

-

6.85

-

6.61

 

-

20.99

6

12.57

-

13.49

-

33.56

-

31.60

-

19.14

-

23.41

-

40.37

-

8

14.91

-

15.94

-

36.46

-

36.46

-

23.62

-

27.18

-

47.13

-

10

15.96

-

18.24

-

41.95

-

39.44

-

26.68

-

32.83

-

52.21

-

14

21.42

-

22.55

-

45.11

-

48.73

-

31.17

-

33.55

-

53.15

-

21

25.58

-

28.39

-

45.74

-

49.91

-

34.81

-

40.33

-

58.92

-

28

35.05

-

35.51

-

60.14

-

57.35

-

44.35

-

51.30

-

66.86

-

29

31.06

2.78

32.84

2.78

57.45

2.78

56.67

2.78

42.53

2.78

50.54

2.78

58.46

2.78

R1 – R2 = Replicates 1 and 2

Abs = CO2 absorber vessels

Table 2: Percentage Biodegradation Values

Day

% Biodegradation

 

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

52

3

25

6

65

28

46

8

70

33

53

10

79

42

59

14

83

35

52

21

69

35

53

28

78

42

53

29

84

49

44

 

Table 3: Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel

Total Carbon* (mg/L)

Inorganic Carbon* (mg/L)

IC Content

(% of TC)

Test Item 10 mg C/L R1

10.18**

0.42

4

Test Item 10 mg C/L R2***

10.14**

-0.58

0

R1 – R2 = Replicates 1 and 2

* Corrected for control values. Negative values are due to measured concentrations being less than control values

** Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

*** Results from analysis of frozen sample

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 49% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. The toxicity control attained 52% biodegradation after 14 days and 44% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. Sodium benzoate attained 83% biodegradation after 14 days and 84% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Description of key information

Not readily biodegradable according to the OECD criteria (49% biodegradation after 28 days, OECD 301 B).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One experimental study is available investigating the biodegradability of the substance. The study was performed according to OECD 301 B (GLP). Non-adapted, domestic activated sludge from a sewage treatment plant was used as inoculum. The test item was found to be not readily biodegradable under the test conditions at the end of the 28 -day exposure period. Thus, the test substance is considered to be “not readily biodegradable” according to the OECD criteria. A toxicity control containing both, reference substance and test substance, did not indicate inhibitory effects to the inoculum (52% degradation after 14 d). All validity criteria of the study were met.