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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:
From 16 December 2009 to 27 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-B (Determination of the "Ready" Biodegradability - Modified OECD Screening Test)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Remarks:
effluent of a sewage treatment plant
Details on inoculum:
- Source of inoculum/activated sludge: effluent from a sewate treatment plant, A-2451 Au am Leithagebirge
- Storage conditions: The effluent was aerated by means of filtered compressed air for about 10 minutes before being used for the study.
- Preparation of inoculum for exposure: The number of cells of the effluent was determined. Each flask was inoculated with 0.5 mL sewage effluent with approximately 3 000 000 cells per mL
- Pretreatment: None.
- Initial cell/biomass concentration: approx. 1 500 000 cells per vessel
Duration of test (contact time):
28 d
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 described in the guidelines
- Additional substrate: None.
- Solubilising agent (type and concentration if used): Not used
- Test temperature: mean temperature: 20.6 °C
- pH: Day 0: 7.4 - 7.5, Day 28: 7.1 - 7.3
- pH adjusted: No.
- Aeration of dilution water: No.
- Suspended solids concentration: Not applicable
- Continuous darkness: Yes.

TEST SYSTEM
- Culturing apparatus: 2L conical flask reaction vessel filled with 1 000 mL test medium.
- Number of culture flasks/concentration: 2 negative control flasks, 2 test substance flask, 1 positive control, 1 toxicity control, 1 abiotic steril control, 1 adsorption control
- Method used to create aerobic conditions: the opening of the vessels was covered with aluminium foil in such a way that the exchang of air was guaranteed.
- Measuring equipment: Measurements were performed with a carbon analyser (TOC-Analysator multi N/C 2000, Analytik Jena).

SAMPLING
- Sampling frequency: Day 0, 2, 4, 7, 10, 14, 17, 21, 24, and 28. Abiotic steril control and adsorption control were determined only on day 28.
- Sampling method: DOC concentrations were determined from the supernatants. At the scheduled terms samples of 30 mL were taken from each reaction vessel and centrifuged (4000 g, 15 min, room temperature).
DOC concentrations were determined from the supernatants at the beginning of the study (Day 0) and then on days 2, 4, 7, 10, 14, 17, 21, 24 and 28 from the groups A, PK, TK and NK. The DOC concentrations of the groups ASK and AK were determined at the beginning of the study and on day 28. The samples from the days 2, 4, 7, 10, 14, 17, 21 and 24 of the groups ASK and AK were stored at -20°C and analysed if appropriate. Measurements were performed with a carbon analyser (TOC-Analysator multi N/C 2000, Analytik Jena, D-07745 Jena, Germany).
Throughout the test concentrations of DOC were determined in samples from each flask in duplicate. The samples were analysed on the same day.
- Sample storage before analysis: Samples were analysed on the same day.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes.
- Toxicity control: Yes.
- Other: Adsorption control

STATISTICAL METHODS: no
Reference substance:
benzoic acid, sodium salt
Preliminary study:
None.
Test performance:
The validity criteria were met.
- Positive control: The reference substance sodium benzoate was degraded by 96.0 % within 14 days.
- Toxicity control: The degradationin the toxicity control, which contained sodium benzoate and the test substance, was not indicative of an inhibition of the microbial activity by the test substance.
- Abiotic control: On Day 0, the DOC concentration was between 18.79 and 18.87 mg/L and on Day 28 between 19.99 and 20.61 mg/L, therefore no abiotic degradation to inorganic carbon occurred in the abiotic sterile control.
- Adsorption control: On Day0, the DOC concentration was between 18.76 and 18.80 mg/L and on Day 28 between 19.56 and 20.39 mg/L, therefore no adsorption of the test substance to the inoculum occurred.
Key result
Parameter:
% degradation (DOC removal)
Value:
11.2
Sampling time:
28 d
Details on results:
The test item was degraded by 11.2 % within 28 days. The test substance is determined to be not ready biodegradable.
The reference substance sodium benzoate was degraded by 96.0 % within 14 days.
Results with reference substance:
The reference substance sodium benzoate was degraded by 96.0 % within 14 days.
The plateau of biodegradation was reached on Day 4 and the degradation of sodium benzoate exceeded the pass level of 70 % within 14 days.

Validity of the test:

The test is considered valid if the difference of extremes of replicate values of the removal of the test substance at the plateau, at the end of the test or at the 10-d window, as appropriate, is less than 20 % and if the percentage degradation of the reference compound has reached the pass levels by day 14.

These criteria were fulfilled.

Percent biodegradation

 

test substance

positive control

toxicity control

Day

A33

A34

mean

PK5

TK36

0

0

0

0

0

0

2

3.48

2.71

3.10

86.61

47.70

4

5.07

5.98

5.52

94.68

52.86

7

5.67

7.04

6.35

95.06

52.52

10

29.97

16.43

23.20

100.17

56.67

14

10.74

10.03

10.38

95.96

53.45

17

- *)

2.49

- *)

98.59

- *)

21

10.82

10.83

10.83

98.03

55.97

24

12.96

11.81

12.39

96.97

57.24

28

17.54

4.80

11.17

99.20

62.83

*)During centrifugation of the day 17 samples the vessels of the individual samples A33 and TK36 broke. Therefore no DOC analysis of these samples could be performed.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The ready biodegradability of the test substance was determined according to OECD 301 E (1992). The test substance was degraded 11.2 % within 28 days of incubation. The test substance is considered to be non- biodegradable.
Executive summary:

Aim and methods

The ready biodegradability of the test substance was determined by the measurement of DOC (dissolved organic carbon) at frequent intervals over a 28 d period. The method used was that described in Part C.4-B of the Regulation (EC) 440/2008 and in the OECD Guideline 301 E.

 

Results

·      The reference substance sodium benzoate was degraded by 96.0 % within 14 days.

·      Degradation in the toxicity control, which contained sodium benzoate and the test substance, was not indicative of an inhibition of the microbial activity by the test substance.

·      The test substance with a nominal starting concentration of 20 mg DOC/L was not degraded within 28 days of incubation at a mean temperature of 20.6 °C.

 

Classification

According to the Guidelines a substance giving a result greater than 70 % loss of DOC in a 10-d window within 28 days is regarded as "readily biodegradable".

According to the results of this study, the test substance can therefore not be classified as readily biodegradable.

Description of key information

The ready biodegradability of the test substance was determined according to OECD 301 E (1992). The test substance was degraded 11.2 % within 28 days of incubation. The test substance is considered to be non- biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability of the test substance was determined by the measurement of DOC (dissolved organic carbon) at frequent intervals over a 28 days period according to OECD 301 E (1992).

The test substance, which provided the sole source of carbon and energy, was dissolved in buffered mineral salts medium at a nominal concentration of about 20 mg organic carbon per litre. The medium wasinoculated with micro-organisms derived from a sample of a sewage effluent from a domestic waste water treatment plant not previously exposed to the test substance. The vessels were incubated in darkness within a specified temperature range for 28 days. The vessels were inserted in a shaking machine and kept there until the end of the study. DOC-determinations were performedin intervals. The test substance was degraded 11.2 % within 28 days of incubation at a mean temperature of 20.6 °C. The test substance is considered to be non- biodegradable.