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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2013-09-21 - 2013-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Qualifier:
according to
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material was used.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge was collected from a municipal wastewater treatment plant (Mannheim, Germany) on 25 Sept 2012 from the aeration tank of the plant.
- Pretreatment: activated sludge suspension was aerated with carbon dioxide free air for about 48 hours at 22 ± 2 °C. At the day of the exposure the
aeration was stopped.
- Concentration of sludge: The sludge of the suspension was adjusted to a concentration of 6.0 g/L of dry weight. Aliquots of 7.5 mL were added
to the test vessels to obtain a sludge concentration of 30 mg/L dry substance in the test assays.
- Water filtered: yes
- Type and size of filter used, if any: sieved by a finely woven mesh (mesh size: 1 mm)
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: TOC
Initial conc.:
33 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: TIC/TOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium complies with test guideline OECD 301B.
- Test temperature: 22 ± 2 °C
- pH adjusted: yes, (final pH of mineral medium = 7.4)

TEST SYSTEM
- Culturing apparatus: 160 mL incubation bottles filled up to a volume of 100 mL (sealed vessels).
- Number of culture flasks/concentration: 2 blank control assays (BC)
3 test substance assays (TS)
1 inhibition control test assays (IH)
1 reference substance assays (RS)
- Measuring equipment: TIC-analysis was performed as repeat determination, using a TOC-analyzer equipped with auto sampler (Shimadzu TOC-5000A).
- Test performed in open system: no

SAMPLING
- Sampling frequency: Samples were taken on day 4, 7, 11, 14, 18, 21, 25 and 28
Reference substance:
aniline
Preliminary study:
None reported.
Test performance:
Neither unusual observations during test nor any other information affecting results are reported.
Parameter:
% degradation (CO2 evolution)
Value:
7
Sampling time:
7 d
Parameter:
% degradation (CO2 evolution)
Value:
27
Sampling time:
11 d
Parameter:
% degradation (CO2 evolution)
Value:
42
Sampling time:
14 d
Parameter:
% degradation (CO2 evolution)
Value:
68
Sampling time:
21 d
Parameter:
% degradation (CO2 evolution)
Value:
75
Sampling time:
28 d
Details on results:
- Duration exposure: 28 d
- Duration of the adaption phase: 8 d
- No inhibition of inoculum by test substance.
- Degree of biodegradation of the test substance at the end of exposure: 70-80% CO2/ThCO2
(ThCO2 = theoretical amount of formed carbon dioxide)
- Degree of biodegradation of the test substance at the end of the ten-day window: not applicable
- Degree of DOC removal of the test substance at the end of exposure: 90-100%
Results with reference substance:
- Degree of biodegradation of the reference substance after 14 d, mean value: 82 % CO2/ThCO2
- Degree of biodegradation in the inhibition control after 28 d, mean value: 73 % CO2/ThCO2

Table 1. Biodegradation degree [CO2/ThCO2]

Test duration

[days]

RS

IH

TS 1

TS 2

TS

mean value

0

0

0

0

0

0

4

36

13

-2

-1

-2

7

59

38

1

12

7

11

76

64

10

43

27

14

82

73

27

56

42

18

86

80

55

64

60

21

89

82

67

69

68

25

90

84

73

72

73

28

91

86

76

74

75

TS= test substance; RS= reference substance; IH= inhibition control

Table 2. DOC values [mg/L]; determined after centrifugation

 

BC 1

BC 2

BC mean value

IH

TS 1

TS 2

TS

mean value

At begin of exposure

a

0.2

0.2

0.3

37.5

19.5

19.3

 

b

0.3

0.5

37.9

19.4

19.4

At end of exposure

a

0.6

0.6

0.6

1.4

1.0

0.9

b

0.6

0.5

1.4

1.2

0.9

Degree of DOC in the test assays [%]

98

97

98

98

TS= test substance; BC= blank control; IH= inhibition control

Validity criteria fulfilled:
yes
Remarks:
Validity criteria of the applied guideline were fulfilled.
Interpretation of results:
other: The substance was readily biodegradable, but the 10-day window was slightly missed based on the mean biodegradation values of both replicates. However, the substance met the criteria for ready biodegradability in the individual assays.
Conclusions:
The study report describes a valid guideline study conducted with certificated GLP compliance. Relevant validity criteria of the test guideline are fulfilled. Butylaminoethanol attained 75 % degradation after 28 days, however, the required degradation degree was not reached within the 10-d window.
Executive summary:

The biodegradability of Butylaminoethanol was investigated using the CO2 Evolution Test in accordance to OECD Guideline 301B (BASF SE, 2013). The selected test concentration (20 mg/L TOC/L) corresponds to approx. 33 mg/L test substance. No toxic effects to the microorganisms were observed, which was confirmed by an additional inhibition control test assay. Aniline was used as reference substance, revealing that the test system is valid. Municipal activated sludge from the wastewater treatment plant was used as inoculum. The TIC- (Total Inorganic Carbon) and DOC (Dissolved Oxygen Carbon)- analyses were performed as repeat determination, using a TOC (Total Organic Carbon) analyzer. The system works with a combustion/non-disperse infrared gas analysis method. The production of carbon dioxide (CO2) is a clear indication of biodegradation. The measured amount of carbon dioxide at the end of the test is compared with the calculated maximal theoretical production (ThCO2) and indicated as biodegradation degree in percent. As the microorganisms oxidize only a part of the test substance and incorporate the rest into biomass, a degradation level > 60 % is considered as sufficient biodegradation. The degree of biodegradation at the end of exposure was measured as 70 - 80 % CO2/ThCO2 (mean value). The pass level for ready biodegradability is the formation of 60 % carbon dioxide compared with the ThCO2 value. This pass level has to be reached in a 10-d window within the 28 day period of the test. The 10-d window begins when the degree of biodegradation has reached 10 % ThCO2 and must end before Day 28 of the test.

Although Butylaminoethanol attained 70 - 80 % degradation, the 10-day window was slightly missed based on the mean biodegradation values of both replicates TS1 and TS2. However, the substance clearly met the criteria for ready biodegradability of chemicals according to the OECD criteria in TS 1 (10-d window: >/=10%: day 11, >/=60%: day 21). The same evaluation applies to TS2 (10-d window: >/=10%: day 7, >/=60%: day 17/18).

Endpoint:
biodegradation in water: screening tests
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2018-03-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
Estimation Programs Interface (EPI) Suite for Microsoft Windows, v4.11 (US EPA, 2012)

2. MODEL (incl. version number)
BIOWIN v4.10

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
See section 'Test Material'.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF.

5. APPLICABILITY DOMAIN
See attached QPRF.

6. ADEQUACY OF THE RESULT
- The model is scientifically valid (see attached QMRF).
- See attached QPRF for reliability assessment.
Principles of method if other than guideline:
The estimation for biodegradability of the test substance was performed with US-EPA software EPIWIN/BIOWIN v4.10. Seven different models are used by this tool to predict the overall biodegradability potential. Anaerobic conditions are considered as well.
GLP compliance:
no
Remarks:
Not applicable.
Specific details on test material used for the study:
CCCCNCCO
Oxygen conditions:
other: aerobic (Biowin 1-6) and anaerobic (Biowin 7)
Inoculum or test system:
other: QSAR estimation
Details on inoculum:
Not applicable.
Details on study design:
Using the computer tool BIOWIN v4.10 by US-EPA (EPIWIN) the aerobic as well as the anaerobic biodegradability of the test material can be estimated. The follwoing seven different models are used by the tool: Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI LInear Model, MITI Non-Linear Model and Anaerobic Model (calles Biowin 1-7, respectively). Due to this results the overall prediction of readily biodegradability is done for the desired chemical.
Reference substance:
other: not applicable
Preliminary study:
Not applicable.
Test performance:
Not applicable.
Remarks on result:
other: Overall Ready Biodegradability Prediction: YES
Details on results:
According to the Linear and also Non-linear Model Butylaminoethanol is biodegrading fast. The Ultimate Biodegradation Timeframe is given in days to weeks, whereas the Primary Biodegradation Timeframe shows weeks for the substance. Both MITI Models predict that the substance is readily biodegradable, which is also the prediction as overall result. Also under anaerobic conditions Butylaminoethanol is suspected to be biodegraded fast.
Results with reference substance:
Not applicable.

Table 1: Biodegradability prediction of Butylaminoethanol

Parameter

Method

Result

Ready Biodegradability Prediction

(withcomputer program from US-EPA (EPIWIN software: BIOWIN Program v4.10))

Biowin1:

Linear Model Prediction

Biodegrades fast

Biowin2:

Non-linear Model Prediction

Biodegrades fast

Biowin3:

Ultimate Biodegradation Timeframe

Days - Weeks

Biowin4:

Primary Biodegradation Timeframe

Days

Biowin5:

MITI Linear Model Prediction

Readily degradable

Biowin6:

MITI Non-Linear Model Prediction

Readily degradable

Biowin7:

Anaerobic Model Prediction

Biodegrades fast

Ready Biodegradability Prediction

YES

Validity criteria fulfilled:
yes
Remarks:
scientifically accepted calculation method
Interpretation of results:
readily biodegradable
Conclusions:
The study report describes a scientifically accepted calculation method for the biodegradability prediction using the US-EPA software BIOWIN v4.10.No GLP criteria are applicable for the usage of this tool and the QSAR estimation is easily repeatable.
Executive summary:

The prediction for biodegradability of the substance Butylaminoethanol was determined by the computer program BIOWIN v4.10 (EPIWIN software) by US-EPA (Chemservice S.A., 2018). The program calculates with seven different models: Linear Model (Biowin 1), Non-linear Model (Biowin 2), Ultimate Biodegradation Timeframe (Biowin 3), Primary Biodegradation Timeframe (Biowin 4), MITI Linear Model (Biowin 5), MITI Non-linear Model (Biowin 6) and Anaerobic Model (Biowin 7). The overall result gives the ready biodegradability prediction of the compound. According to Biowin 1 and 2 the substance is biodegrading fast. The Ultimate Biodegradation Timeframe is given in days to weeks, whereas the Primary Biodegradation Timeframe gives days as result. Both MITI Models predict that the substance is readily biodegradable, which is also the overal prediction result. Also under anaerobic conditions the substance is expected to be degraded fast.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-05-06 to 2011-07-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material was used.
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Secondary effluent of a predominantely domestic WWTP;
Location: Cambridge STP, Cowley Road, Cambridge, UK.
Date of collection: 2001-06-30.
- Laboratory culture: No
- Pretreatment: Filtered (Whatman No. 54)
- Concentration of sludge: 1 mL/L
- Initial cell/biomass concentration: Not reported
- Water filtered: yes
- Type and size of filter used, if any: Whatman No. 54
Duration of test (contact time):
28 d
Initial conc.:
8.61 mg/L
Based on:
DOC
Initial conc.:
2.79 mg/L
Based on:
test mat.
Initial conc.:
2 325 other: mg O2/g
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
other: BOD and ThOD was calculated
Details on study design:
TEST CONDITIONS
- Composition of medium: addition of sample stock solutions to inoculated mineral medium.
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 20.3 to 20.3°C
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: standard BOD bottles (closed filled bottles)
- Number of culture flasks/concentration: two
- Measuring equipment: dissolved oxygen meter
- Test performed in closed vessels due to significant volatility of test substance: no

SAMPLING
- Sampling frequency: 0, 7, 14, 21 and 28 days after inoculation
- Sampling method: measurement of DO (dissolved oxygen) concentration
- Sterility check if applicable: not reported
- Sample storage before analysis: no storage before sampling

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (inoculated mineral medium only)
- Abiotic sterile control: none
- Toxicity control: reference substance: sodium acetate

STATISTICAL METHODS: None
Reference substance:
acetic acid, sodium salt
Preliminary study:
No preliminary study reported.
Test performance:
Neither unusual observations during test nor any other information affecting results are reported.
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
26
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
27
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
61
Sampling time:
28 d
Details on results:
No further details are reported.
Results with reference substance:
71 % degradation after 7 days,
72 % degradation after 14 days,
76 % degradation after 21 days,
75 % degradation after 28 days.

3.1 Biochemical oxygen demand

 

Incubation period (days)

 

0

7

14

21

28

Incubator temperature (°C)

20.0

20.2

20.2

20.3

20.3

Average blank - DO (mg/L)

8.65

8.43

8.23

7.92

7.59

Replicate number

1

1

2

1

2

1

2

3

1

2

3

Test substance - DO (mg/L)

8.61

8.44

8.29

8.08

4.91

6.12

6.29

5.95

3.70

3.41

3.58

Test substance - BOD (mg/L)

-

-18

36

39

1176

631

570

692

1380

1484

1423

Reference material - DO (mg/L)

8.61

5.34

5.51

5.49

4.87

4.82

4.85

4.48

4.50

4.43

4.27

Reference material - BOD (mg O2/g)

-

568

536

503

618

570

564

633

568

581

611

 

3.3 Degradation

 

Incubation period (days)

 

7

14

21

28

Replicate number

1

2

1

2

1

2

3

1

2

3

Test substance (%)

-1

2

2

51

27

25

30

59

64

61

Average degradation (%)

0

26

27

61

Reference material (%)

73

69

64

79

73

72

81

73

74

78

Average degradation (%)

71

72

76

75

 

Validity criteria fulfilled:
yes
Remarks:
Validity criteria of the applied guideline are fulfilled.
Interpretation of results:
readily biodegradable
Conclusions:
The study report describes a valid guideline study conducted with certificated GLP compliance. Relevant validity criteria of the test guideline are fulfilled. Butylaminoethanol attained 61 % degradation after 28 days, thus is considered as readily biodegradable.
Executive summary:

A ready biogradability test was performed with Butylaminoethanol using the closed bottle test according to OECD Guideline 301 D (Drake, 2011). Predominantely domestic activated sludge was used as inoculum (inoculum concentration 1 mL/L). Incubation period was 28 days and degradation was followed by analysis of the dissolved oxygen. After 7, 14, 21 and 28 days of incubation degradation rates were detected for the test substance as well as for the reference substance (positice control) sodium acetate. The calculated ThOD was 2325 mg O2/g test substance. Following degradation rates were observed after 7, 14, 21 and 28 days: 0, 26, 27, 61 %, respectively. The reference substance showed rapid degradation: 71 % after 7 days and 75 % after 28 days, respectively.

Description of key information

Ready biodegradation_OECD 301 D: 61 % degradation after 28 days - readily biodegradable,
Ready biodegradation_OECD 301 B: 75 % degradation after 28 days - readily biodegradable, but failing 10-day window (based on mean values of replicates; the substance turned out to be readily biodegradable in the individual assays),
Ready biodegradation_BIOWIN v4.10_overall prediction: Yes, readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Two experimental results as well as one QSAR prediction are available regarding biodegradability of Butylaminoethanol. As key information serves a ready biodegradability study performed with Butylaminoethanol using the closed bottle method according to OECD Guideline 301 D (Drake, 2011). Predominantly domestic activated sludge was used as inoculum (inoculum concentration 1 mL/L). Incubation period was 28 days and degradation was followed by analysis of the dissolved oxygen. After 7, 14, 21 and 28 days of incubation degradation rates were detected for the test substance as well as for the reference substance (positive control) sodium acetate. The calculated ThOD was 2325 mg O2/g test substance. Following degradation rates were observed after 7, 14, 21 and 28 days: 0, 26, 27, 61 %, respectively. The reference substance showed rapid degradation: 71 % after 7 days and 75 % after 28 days, respectively. Based on these test results, Butylaminoethanol can be considered as readily biodegradable.

 

In a newly conducted CO2 Evolution Test which was performed according to OECD Guideline 301B is reported (BASF SE, 2013). The selected test concentration (20 mg/L TOC/L) corresponds to approx. 33 mg/L test substance. No toxic effects to the microorganisms were observed, which was confirmed by an additional inhibition control test assay. Aniline was used as reference substance, revealing that the test system is valid. Municipal activated sludge from the wastewater treatment plant was used as inoculum. The TIC- (Total Inorganic Carbon) and DOC (Dissolved Oxygen Carbon) - analyses were performed as repeat determination, using a TOC (Total Organic Carbon) analyzer. The system works with a combustion/non-disperse infrared gas analysis method. The production of carbon dioxide (CO2) is a clear indication of biodegradation. The measured amount of carbon dioxide at the end of the test is compared with the calculated maximal theoretical production (ThCO2) and indicated as biodegradation degree in percent. As the microorganisms oxidize only a part of the test substance and incorporate the rest into biomass, a degradation level > 60 % is considered as sufficient biodegradation. The degree of biodegradation at the end of exposure was measured as 70 - 80 % CO2/ThCO2 (mean value: 75 %). The pass level for ready biodegradability is the formation of 60 % carbon dioxide compared with the ThCO2 value. This pass level has to be reached in a 10-d window within the 28 day period of the test. The 10-d window begins when the degree of biodegradation has reached 10 % ThCO2 and must end before Day 28 of the test. However, Butylaminoethanol slightly missed this 10-d window taking into account the mean biodegradation values of the replicates TS1 and TS2. However, the substance clearly met the criteria for ready biodegradability of chemicals according to the OECD criteria in TS1: 10-d window: >/=10%: day 11, >/=60%: day 21. The same evaluation applies to TS2 (10-d window: >/=10%: day 7, >/=60%: day 17/18).

 

A supporting QSAR prediction was performed with BIOWIN v4.10 (EPIWIN software) by US-EPA (Chemservice S.A., 2018). The program calculates with seven different models: Linear Model, Non-linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model, and Anaerobic Model (called Biowin 1 – 7, respectively). Due to the overall results a prediction of the ready biodegradability is done for the chemical. This can only be predicted for organic substances.If the Biowin 3 result is given in weeks or even faster and the Biowin 5 probability is ≥ 0.5, then the overall prediction is “yes”, thus the substance will be considered as readily biodegradable. This procedure is based on application of Bayesian analysis to ready biodegradation data. Biowin 5 and 6 predict the ready biodegradability for degradation in the test system of OECD Guideline 301C only and thereby using the database from the Chemicals Evaluation and Research Institute Japan (CERIJ). According to Biowin 1 and 2 the substance is biodegrading fast. The Ultimate Biodegradation Timeframe is given in days to weeks, whereas the Primary Biodegradation Timeframe gives days as result. Both MITI Models predict that the substance is readily biodegradable, which is also the overall prediction result. Also under anaerobic conditions the substance is expected to be degraded fast.

 

Taking into account all available information, Butylaminoethanol can be considered as readily biodegradable.