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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:
12 Jul - 26 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
Temperature range was 19.4 – 21.6 °C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range, this is considered as uncritical concerning the outcome of the study.
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
2008
Deviations:
yes
Remarks:
Temperature range was 19.4 – 21.6 °C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range, this is considered as uncritical concerning the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf, Germany; Date of collection: 25. Aug. 2017
- Preparation of inoculum for exposure: Inoculum was taken from its source, washed, aerated and the dry matter was determined; test vessels were filled with medium and inoculum and aerated afterwards.
- Pretreatment: Sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use for 96 hours with purified, CO2-free, moistened air to purge the system of CO2.
- Concentration of sludge: 4120 mg suspended solids/L
- Inoculum concentration: 25.0 mg/L dry matter/L
Duration of test (contact time):
28 d
Initial conc.:
29 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
other: organic C
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to the OECD guideline 301
- Test temperature: 19.4 – 21.6 °C (see deviation in section „Any other information on material and methods incl. tables“)
- Suspended solids concentration: Dry matter was determined as 4600 mg suspended solids/L.
- Continuous darkness: incubated in the dark
- pH: 6.9 - 7.5

TEST SYSTEM
- Culturing apparatus: 2000 mL-SCHOTT-flasks (filled with 1500 mL test solution) were used as test vessels, 100 mL scrubber flasks as absorbent vessels.
- Number of culture flasks/concentration: 2 (per test item, blank, apparatus blank and reference) and 1 reactor (per toxicity control and abiotic control)
- Method used to create aerobic conditions: The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels. Magnetic stirrers were used to prevent deposition of inoculum.

- Measuring equipment: Analysis of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon analyser was calibrated with freshly prepared reference solutions containing potassium hydrogen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC) every month. After every start, quality control samples were measured.

- Details of trap for CO2 and volatile organics if used: The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.

SAMPLING
- Sampling frequency: On the 0, 2nd, 4th, 7th, 9th, 11th, 14th, 18th, 23rd and 29th day
- Sampling method: From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2. The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2. On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.

CONTROL AND BLANK SYSTEM
- Apparatus blanks: yes, 2 reactors (containing mineral medium only)
- Inoculum blank: yes, 2 reactors (containing mineral medium and inoculum)
- Toxicity control: yes, 1 reactor (containing test item, positive control, mineral medium and inoculum)
- Reference control: yes, 2 reactors (containing positive control, mineral medium and inoculum)
- Abiotic control: yes, 1 reactor (containing test item, mineral medium and HgCl2)

Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
84
Sampling time:
28 d
Results with reference substance:
Degradation of the positive control was 69.7% after 9 days.

Table 1: Degradation values in %

Day

Positive Control 1

Positive Control 2

Positive Control Mean

Test 1

Test 2

Test Mean

Abiotic Control

Toxicity Control

2

0.4

-3.2

-1.4

10.0

22.9

16.5

0.3

10.7

4

16.0

8.7

12.4

28.4

36.9

32.7

0.4

43.0

7

52.8

54.2

53.5

49.3

53.7

51.5

0.3

71.9

9

69.0

70.5

69.7

58.6

62.3

60.4

0.2

78.3

11

79.7

78.1

78.9

68.3

69.4

68.9

0.0

79.5

14

84.3

79.4

81.9

56.7

74.3

65.5

0.7

80.3

18

88.5

80.7

84.6

79.4

77.4

78.4

-0.3

84.0

23

84.8

78.5

81.7

78.8

79.1

78.9

-1.0

82.7

29

93.1

84.5

88.8

84.5

83.9

84.2

-1.4

88.2

 

Table 2: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

0.7%

Yes

Percentage degradation of the reference compound has reached the pass levels by day 14.

9 d

Yes

The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC, and the total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.

0% and 11.5 mg/L

Yes

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
readily biodegradable

Description of key information

Readily biodegradable: 84% within 28d (OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

One GLP guideline study according to OECD 301B is available investigating the ready biodegradability of Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate under aerobic conditions using domestic activated sludge as inoculum. A mean biodegradation of 84% was observed after 28 d. The substance reached the pass level biodegradation of > 60% within the 10-day window and therefore considered as readily biodegradable.