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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:
2017-02-22 to 2017-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- TOC value of the test substance: 666 mg/g
- Purity: 99.2 area %
- Batch Nr.:85603856P0
- Homogeneity: homogeneous
- Physical state / appearance: liquid / colorless, clear
- Density: 0,8168 g/mL at 23 °C
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany was collected on 27 February 2017 from the aeration tank of the plant. A suitable aliquot of the suspension was sieved by a finely woven mesh with a mesh size of about 1 mm. To reduce the content of inorganic carbon in the blank control the activated sludge suspension was aerated with pressured air for about 48 hours at 22 ± 2° C. At the day of exposure, the aeration was stopped and the sludge was allowed to settle. The supernatant was discarded and the residue of the sludge suspension was filled up with drinking water. The sludge of the suspension was adjusted to a concentration 800 mg/L of dry weight. Aliquots of 0.5 mL were added to the test vessels to obtain a sludge
concentration of 4 mg/L dry substance in the test assays. Reason for the choice of the test system: The test guideline recommends the usage of activated sludge from a wastewater treatment plant, treating municipal sewage.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
29 blank control assays
31 test substance assays
29 inhibition control test assays
17 reference substance assays
5 assays for abiotic control

The pH value of the mineral medium was checked and adjusted to 7.4 with 1 molar sulphuric acid. The CO2-Headspace Test was performed in 160 mL incubation bottles filled up to a volume of 100 mL. The oxygen, available in the headspace of the bottles, is used for the aerobic mineralization processes. The amount of oxygen allows the complete biodegradation. The aqueous phase, a mineral medium with municipal inoculum contains the test compound, directly added by a suitable micro syringe. The bottles were closed with butyl rubber septa gas tight and shaken by an orbital shaker with a speed of about 150 r/min and incubated at a temperature of 22 ± 2 °C.
Reference substance:
aniline
Preliminary study:
Yes. A pH- shift in the test vessels, induced by addition of the test substance to the test medium was checked in a preliminary test. No shift of the pH-value was detected. The pH-value from the mineral test medium was therefore assumed for the test
assays at start of exposure. This pH-value of the mineral medium after correction was 7.4.
Test performance:
All validity criteria were met and no deviations from the test guidelines or other incidents occurred during the course of the reported test which may have influenced.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
28 d
Results with reference substance:
- biodegradation: 82 % after 14 days

Nominal TOC [mg/L] concentration in the test assays: RS : 20.0, IH: 40.0, PC: 20.0, TS: 20.0. Abbreviations: TS = test substance assay, RS = reference substance assay, IH = inhibition control assay, PC = abiotic control assay, TOC = total organic carbon, mv = mean value.

 

Degree of biodegradation [%]

Test duration [days]

RS1

RS2

RS3

R mv

IH1

IH2

IH3

IH mv

TS1

TS2

TS3

TS mv

2

2

2

2

2

2

4

1

2

0

1

1

1

5

57

58

58

58

28

28

28

28

0

0

-1

0

8

76

78

75

76

42

38

40

40

1

1

1

1

12

81

79

81

80

40

39

38

39

0

0

-1

0

14

84

83

79

82

39

43

43

42

1

1

1

1

19

40

40

40

40

1

0

0

0

23

42

43

45

43

0

0

1

0

26

43

42

42

42

0

0

0

0

Test duration [days]

PC1

PC2

PC3

P mv

IH1

IH2

IH3

IH mv

TS1

TS2

TS3

TS4

28

-1

-1

-2

-1

40

40

42

41

-1

0

0

1

TS5

TS mv

0

0

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable

Description of key information

not readily biodegradable

Key value for chemical safety assessment

Additional information

The ready biodegradability of the test substance was investigated under GLP conditions by means of a CO2 Headspace Test, according to OECD guideline 310. The biodegradation of the test substance was followed by exposing it to microorganisms of non-adapted activated sludge from a municipal wastewater treatment plant. An inoculum control, containing sole pretreated activated sludge, a functional control with the reference substance aniline as well as a toxicity control, including both test item and reference item, was conducted simultaneously. The test concentration selected as appropriate was 20 mg/L TOC, corresponding to approximately 30 mg/L test substance. The flasks were incubated for 28 days at 22 ± 2 °C under aerobic and dark conditions. The biodegradability was derived from TIC analysis of the quantity of CO2 produced within the test period by respiration of bacteria. The mean value of degree of biodegradation was calculated by the single values of five test assays at the end of exposure with a limit of confidence of 95 %.

In result, the required pass level for ready biodegradability was not reached. The degree of biodegradation after an exposure period of 28 days was 0 ± 1 % TIC/TOC. As proven by the toxicity control, no inhibitory effects on microorganisms is expected at the tested concentration of 20 mg/L (degree of biodegradation after 28 days: 41 %).

In conclusion, the test item is not regarded to be in the 10-d-window and after 28 days readily biodegradable. Based on determined rate of biodegradation at the end of exposure the test item can be evaluated as poorly biodegradable in this test. All validity criteria were met and no deviations from the test guidelines or other incidents occurred during the course of the reported test which may have influenced. Based on this result, the test substance has to be regarded as not readily biodegradable.