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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:
01 Jun - 07 Jul 2017
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:
adopted 17. Jul. 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
30. May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Mainz, Germany
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Biological sewage treatment plant; sludge from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, NW-Lachen-Speyerdorf, Germany (Date of collection: 02 Jun 2017, batch no: 20170602)
- Storage conditions: The sludge was aerated until use.
- Inoculum concentration: 25.0 mg/L
- Pretreatment: The sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium.
- Concentration of sludge: The dry matter was determined as 4840 mg suspended solids/L.
- Other: All flasks containing inoculum and medium were aerated for 96 h with purified, CO2-free, moistened air to purge the system of CO2.
Duration of test (contact time):
28 d
Initial conc.:
>= 19.9 - <= 20.1 mg/L
Based on:
other: organic carbon/L
Initial conc.:
>= 27.7 - <= 27.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: According to the guideline 301
- Test temperature: 20.0 – 21.3 °C
- pH: 7.9 (blank control); 7.7 (positive control); 7.9 (test item); 7.3 (abiotic control); 7.8 (toxicity control) at the end of the test, on day 28
- Suspended solids concentration: 4840 mg suspended solids/L.

TEST SYSTEM
- Culturing apparatus: 2000 mL-SCHOTT-flasks were used as test vessels, 100 mL scrubber flasks as absorbent vessels.
- Number of culture flasks/concentration: 2 (per test item, blank and reference) and 1 reactor (toxicity control and abiotic control)
- Method used to create aerobic conditions: Test vessels were filled with inoculum and medium and were aerated for 96 h with purified, CO2-free, moistened air to purge the system of CO2. 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.
- Measuring equipment: IC measurement was performed 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.
- Test performed in open system: Yes
- Details of trap for CO2 and volatile organics if used: Emitted CO2 was trapped in 0.25 M NaOH; two scrubbers containing 100 mL each were connected in series to the test vessels; initial IC value of the 0.25 M NaOH was separately determined in each flask.

SAMPLING
- Sampling frequency: Test start and on the 2nd, 6th, 8th, 10th, 14th, 17th, 23rd and 29th day
- Sampling method: From each front scrubber flask, 9 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 blank: yes, 2 bottles (containing mineral medium only)
- Inoculum blank: yes, 2 bottles
- Abiotic sterile control: yes, 1 bottle
- Toxicity control: yes, 1 bottle
- Reference control: yes, 2 bottles
Reference substance:
aniline
Remarks:
26.3 mg/L
Parameter:
% degradation (CO2 evolution)
Value:
11
Sampling time:
28 d
Remarks on result:
other: Mean of two replicates
Details on results:
Other: Results in details are summarized within the tables 1-3 in section "Any other information on results incl. tables"
Results with reference substance:
Degradation of the positive control was 68% after 8 days.

The degree of biodegradation reached 11% after 28 days. The criterion for ready biodegradation (degradation 60%, 10-d-window) was therefore not met.

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

-1.1

-0.4

-0.7

-0.7

1.1

0.2

1.1

0.1

6

33.4

64.9

49.1

0.9

3.5

2.2

-0.4

32.5

8

51.2

84.1

67.6

1.7

3.9

2.8

-0.8

42.0

10

62.6

87.1

74.9

2.1

3.6

2.9

-1.1

44.5

14

78.7

92.1

85.4

1.8

5.2

3.5

-1.2

46.3

17

81.3

89.0

85.2

2.7

6.6

4.7

-1.4

44.5

23

85.3

91.2

88.3

5.1

7.3

6.2

-1.7

46.7

29

88.6

89.5

89.1

12.8

9.6

11.2

-2.7

45.8

 

Table 2: Emitted carbon in mg/L

Day

Blank Control 1

Blank Control 2

Positive Control 1

Positive Control 2

Test 1

Test 2

Abiotic Control

Toxicity Control

2

0.32

0.38

0.13

0.27

0.20

0.56

0.23

0.40

6

0.28

0.82

7.24

13.55

0.74

1.24

-0.08

13.60

8

0.54

1.09

11.08

17.66

1.16

1.59

-0.17

17.66

10

0.86

1.49

13.72

18.62

1.59

1.90

-0.22

19.02

14

1.34

2.19

17.52

20.21

2.12

2.79

-0.24

20.36

17

1.31

2.26

18.07

19.63

2.33

3.11

-0.28

19.65

23

1.79

2.65

19.31

20.50

3.25

3.68

-0.35

20.97

29

2.42

2.73

20.32

20.51

5.15

4.50

-0.54

20.94

Table 3: Validity criteria

Parameter

Criterion

Found

Assessment

IC content of test item solution in medium

≤ 5% of TC

0%

valid

CO2 emitted by the controls

< 70 mg/L

9.4 mg/L

valid

Difference within replicates

≤20%

3.2%

valid

Degradation of positive control > 60%

≤ 14 days

8 days

valid

Degradation in the toxicity flask on day 14

> 25%

46.3%

valid

Validity criteria fulfilled:
yes
Remarks:
For detailed information please see section "Any other information on results incl tables"
Interpretation of results:
not readily biodegradable
Conclusions:
(2Z)-4-(octadecylamino)-4-oxo2-butenoic acid is not readily biodegradable and not ultimately biodegradable following OECD 301B and EU C.4-C, respectively.

Description of key information

Not readily biodegradable (11% after 28 d, OECD 301 B)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

There is one GLP study available, which investigated the ready biodegradability of the test substance according to OECD guideline 301 B. In a CO2 evolution test, nominal test item concentrations of 27.7 and 27.9 mg/L were inoculated with 25 mg/L dry matter of microorganisms from activated sludge of a municipal sewage treatment plant in glass bottles under static exposure conditions for 28 d. Degradation was followed by the measurement of CO2 evolution. After 28 d, degradation of the test item reached 11%. Therefore, the test item did not reach the criteria for ready biodegradability. The reference control confirmed the suitability of the test system. Degradation in the toxicity control was 46.3% after 14 d. Thus, the test item is not toxic to microorganisms of activated sludge according to the guideline criteria.