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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-16 to 2016-04-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
440/2008/EEC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Sewage treatment plant Ruhrverband Kläranlage, Schmallenberg, mainly fed with municipal wastewater
- Concentration: 29.5 mg dry mass/L
Duration of test (contact time):
28 d
Initial conc.:
60 mg/L
Based on:
test mat.
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
(a) KH2PO4: 8.5 g/L
K2HPO4: 21.75 g/L
Na2HPO4: 24.93 g/L
NH4Cl: 0.50 g/L
pH 7.4 +/- 0.2
(b) CaCl2 x 2H2O: 36.40 g/L
(c) MgSO4 x 7 H2O: 22.50 g/L
(d) FeCl3 x 6 H2O: 0.25 g/L
The mineral medium applied in the test contained 10 mL/L of mineral stock solution (a) and 1 mL/L of the mineral stock solutions (b) - (d), respectively.
- Solubilising agent (type and concentration if used): none
- Test temperature: 22 °C (+/-) 1 °C
- pH: 7.5-7.9
- pH adjusted: no
- Continuous darkness: yes
TEST SYSTEM
- Number of culture flasks/concentration: Two vessels/concentration
- Method used to create aerobic conditions: Aeration during the whole test
- Measuring equipment: SAPROMAT respirometer (VOITH Inc.)
- Test performed in closed vessels
SAMPLING
- Sampling frequency: continuous measurement of the oxygen demand using a SAPROMAT respirometer
CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: 100 mg test item/L sterilised mineral test medium (25mg/250 mL)
- Toxicity control: 100 mg test item/L and reference item at 100 mg/L mineral test medium (25 mg test item/250 mL and 25 mg reference item/250 ml)

Reference substance:
benzoic acid, sodium salt
Test performance:
The test item in concentrations of about 100 and 60 mg per litre mineral medium, respectively, and sodium benzoate in concentrations of about 100 mg per litre mineral medium, were incubated with 29.5 mg dry mass inoculum per litre mineral medium in 500 mL glass vessels at a medium volume of 250 mL. The test was run for 28 days, in darkness at 22 °C (+/-) 1 °C. The suspension was aerated during the whole test. Oxygen demand was carried out throughout the test course.
Parameter:
% degradation (O2 consumption)
Value:
69
St. dev.:
0.8
Remarks on result:
other: continous measurement
Parameter:
% degradation (O2 consumption)
Value:
85
St. dev.:
5.4
Remarks on result:
other: continous measurement
Details on results:
The biodegradation within the 10-day-window was 61 % and 75 % in the assays with 100 mg/L and 60 mg/L, respectively. The 10-day-window started at day 1 for a concentration of 60 mg/L and at day 2 for a concentration of 100 mg/L
Results with reference substance:
The degradation of the reference substance sodium benzoate had reached 83 % within the first 14 days.

The biodegradation of the test item after 28 days of incubation in the static test was found to be 69 % (SD = 0.8 %) and 85 % (SD = 5.4 %) in the assays with 100 mg/L and 60 mg/L, respectively. The biodegradation within the 10-day-window was 61 % and 75 % in the assays with 100 mg/L and 60 mg/L, respectively. The 10-day-window started at day 1 for a concentration of 60 mg test item per litre and at day 2 for a concentration of 100 mg test item per litre.

With 4 %, there was no significant abiotic degradation of the test item noticeable within the 28 days of incubation.

The biodegradation of the item mixture in the toxicity control was found to be 65 % after 14 days of incubation. Thus, the demanded threshold value of 25 % is exceeded and the test item can be identified as non-toxic in a ready biodegradability test.

The reference item sodium benzoate was degraded to 83 % within the first 14 days.

Table 1: pH values at test start. Single values of the parallel test vessels.

vessel

Inoculum blank

Test suspension A

Test suspension B

Abiotic control

Procedural control

Toxicity control

1

7.5

7.5

7.5

7.5

7.5

7.5

2

7.6

7.5

7.5

7.5

7.5

7.5

Table 2: pH values at test end. Single values of the parallel test vessels.

vessel

Inoculum blank

Test suspension A

Test suspension B

Abiotic control

Procedural control

Toxicity control

1

7.7

7.9

7.9

7.7

8.5

8.8

2

7.8

7.9

7.9

7.7

8.6

8.8

Table 3: Oxygen consumption. Cumulated consumption (mg O2/L) after 14 days. Single and mean values of the parallel test vessels and standard deviation. Test suspension B: 60 mg/L; Test suspension A and abiotic control: 100 mg/L; Procedural control: 100 mg/L; Toxicity control: 200 mg/L.

vessel

Inoculum blank

Test suspension A

Test suspension B

Abiotic control

Procedural control

Toxicity control

1

22

76

59

0

160

189

2

23

77

67

3

161

187

Mean

23

77

63

2

161

188

SD

1

1

6

2

1

1

Table 4: Percent degradation. Degradation (%) after 14 days. Single and mean values of the parallel test vessels and standard deviation.

vessel

-

Test suspension A

Test suspension B

Abiotic control

Procedural control

Toxicity control

1

-

61.8

70.3

0.0

82.6

65.8

2

-

63.0

85.7

3.5

83.2

65.0

Mean

-

62.4

78.0

1.7

82.9

65.4

SD

-

0.8

10.9

2.5

0.4

0.6

Table 5: Per Oxygen consumption. Cumulated consumption (mg O2/L) after 28 days. Single and mean values of the parallel test vessels and standard deviation. Test suspension B: 60 mg/L; Test suspension A and abiotic control: 100 mg/L; Procedural control: 100 mg/L; Toxicity control: 200 mg/L. cent degradation. Degradation (%) after 14 days. Single and mean values of the parallel test vessels and standard deviation.

vessel

Inoculum blank

Test suspension A

Test suspension B

Abiotic control

Procedural control

Toxicity control

1

28

88

70

0

172

204

2

28

87

74

7

174

205

Mean

28

88

72

4

173

205

SD

0

1

3

5

1

1

Table 6: Percent degradation. Degradation (%) after 28 days. Single and mean values of the parallel test vessels and standard deviation.

vessel

-

Test suspension A

Test suspension B

Abiotic control

Procedural control

Toxicity control

1

-

69.3

80.9

0.0

86.5

69.5

2

-

68.2

88.6

8.1

87.7

69.9

Mean

-

68.7

84.7

4.0

87.1

69.7

SD

-

0.8

5.4

5.7

0.8

0.3

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The biodegradation of the test substance after 28 days incubation in the static test was found to be 69 % and 85 % in the assays with 100 mg/L and 60 mg/L, respectively.
Executive summary:

The biodegradation of the test substance was investigated over a 28 day period in a manometric Respirometry Test according to OECD Guideline 301 F (1992). The test medium was inoculated with microorganisms from a digester of a sewage treatment plant mainly fed with municipal wastewater.

The rate of degradation was monitored by measuring the quantity of oxygen required to maintain a constant gas volume in the respirometer flasks over 28 days.

Sodium benzoate was used as a degradable reference substance at a concentration of 100 mg/L, along with a toxicity control at 100 mg/L test substance and 100 mg/L sodium benzoate.

The biodegradation of the test substance was found to be at mean 69 % with a standard deviation of 0.8 % for a concentration of 100 mg test substance/L and at mean 85 % with a standard deviation of 5.4 % for a concentration of 60 mg test item/L. For a concentration of 100 mg test substance/L biodegradation within the 10-day-window was found to be 61 %.

For a concentration of 60 mg test substance/L biodegradation within the 10-day-window was found to be 75 %.

The degradation of of the reference substance sodium benzoate had reached 83 % within the first 14 days.

According to the guideline, the test item is considered as being readily biodegradable under the test conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009 2009-02-19 to 2009-11-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 311 (Anaerobic Biodegradability of Organic Compounds in Digested Sludge: Measurement of Gas Production)
Version / remarks:
adopted 23 March 2006
Deviations:
yes
Remarks:
Several minor deviations which were considered irrelevant or uncritical for the result of the study
Qualifier:
according to
Guideline:
other: EN ISO 11734
Version / remarks:
1998
Deviations:
yes
Remarks:
Several minor deviations which were considered irrelevant or uncritical for the result of the study
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
anaerobic
Inoculum or test system:
anaerobic sludge
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Digester of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lache-Speyerdorf; batch number 25092009
- Pretreatment: The sludge was centrifuged and washed with anoxic test medium, then ventilated with oxygen-free Ar and re-suspended in anoxic test medium. A pre-fermentation was performed at 37 °C for five days. Sufficient washing was performed to reduce the content of inorganic carbon to < 12 mg/L. The sludge was diluted to give a concentration of solid in the tests of 3 g/L. Before tzhe start of the test the sludge was again ventilated with Ar.
Duration of test (contact time):
61 d
Initial conc.:
397.7 mg/L
Based on:
test mat.
Initial conc.:
172.4 mg/L
Based on:
act. ingr.
Initial conc.:
407.7 mg/L
Based on:
test mat.
Initial conc.:
176.8 mg/L
Based on:
act. ingr.
Initial conc.:
396 mg/L
Based on:
test mat.
Initial conc.:
171.7 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
other: Pressure of developed gas
Details on study design:
TEST CONDITIONS
- Composition of medium:
Potassium dihydrogene phosphate: 0.27 g
di-Sodium hydrogene phosphate-dihydrate: 0.555 g
Ammonium chloride: 0.53 g
Magnesium chloride hexahydrate: 0.1 g
Stock solution of trace elements: 10 mL
Iron-(II)-chloride tetrahydrate: 0.02 g
Resazurin (indicator for oxygen): 0.001 g
Sodiu sulfide: 0.1 g
deionised water: ad 1000 mL
- Test temperature: 35 (+/- 2) °C
- pH: 7.0 (+/- 0.2)
- pH adjusted: yes

TEST SYSTEM
-Inoculum concentration: 3 g suspended solids/L
- Volume of flasks: 500 mL
- Control flasks: 3 (medium and inoculum)
- Reference flasks: 3 (phenol, medium and inoculum)
- Test flasks: 3 (test substance, medium and inoculum)
- Toxicity control: 3 (test substance, phenol, medium and inoculum)
- Method used to create anaerobic conditions: The medium was prepared and purged with Ar. The gas was purged from oxygen using three washing bottles (first bottle with dithionite as oxygen acceptor, second bottle with pyrogallol as oxygen indicator, third bottle as liquid trap) The inoculum was taken from its source and prepared as described under "Details on inoculum".
On the day of the start of the test the stock solution for the positive control as well as fresh test medium were prepared and ventilated with Ar. The inoculum was diluted with medium to a concentration of solids of 3 g/L.
- Measuring equipment:
500 mL glass flakes with screw caps
OxyTop OC110 and measuring heads OxiTop-C

SAMPLING
No samples were taken; instead the pressure of the evolved gas in each flask was measured continously until the end of the test.
CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

Reference substance:
other: Phenol
Key result
Parameter:
other: Pressure of developed gas
Value:
110.7
Remarks on result:
other: Mean value from three vessels
Details on results:
The degree of biodegradation reached 100 %.
Lag-Phase: after 3 days, significant increase of pressure was detectable
Plateau-Phase: after 40 days, no significant pressure increase could be observed
Degradation at the end of the test: 100 % (calculated value 111 %)
The calculated degradation at the end of the test was more than 100 %. This might be due to the following: in the test flasks, bacterial growth was enhanced by the well biodegradable test item. After digestion of the test item, the biomass started self-digestion as no other organic nutrients were available. Self-digestion of the previously increased biomass in the test flasks might very well been higher than in the control flasks.

Carbon content in the gas phase at the end of the test

Emitted carbon in the gas phase in mg was calculated using the following equation:

Mh= 0.468 x Δpkum x Vh

with

mh=     net weight of emitted carbon in the gas phase in mg

Δpkum=               cumulated gas pressure test minus mean gas pressure control:

               pT(1) – pBI(1) + pT(2) + …pT(n) – pBI(n)

 

Flask

Pressure

Net Pressure

Gas phase

Net carbon content Gas Phase

Designation

hPa

hPa

L

mg

Control 1

84

 

0.1310

--

Control 2

72

 

0.1192

--

Control 3

54

 

0.1191

--

Control mean

70

 

 

--

Pos. Control 1

90

20

0.1295

1.2

Pos. Control 2

86

16

0.1023

0.8

Pos. Control 3

84

14

0.1101

0.7

Pos. Control Mean

87

17

 

0.9

Test 1

850

780

0.1016

37.1

Test 2

846

776

0.1160

42.1

Test 3

878

808

0.1012

38.3

Test Mean

858

788

 

39.2

Toxicity control 1

771

701

0.1225

40.2

Toxicity control 2

871

801

0.1251

46.9

Toxicity control 3

953

883

0.1271

52.5

Toxicity control Mean

865

795

 

46.5

 

Inorganic carbon content in the liquid phase in mg

The carbon content in the liquid phase is is calculated using the following equation:

mi = IC(Test) x Vf(Test) – Σ3i-1[IC(Control)I x Vf(Control)i]/3

with

mi =                                                                     inorganic carbon content in the liquid phase in mg

IC(Test) x Vf(Test) =                                     inorganic carbon in the respective test flask in mg

Σ3i-1[IC(Control)I x Vf(Control)i]/3 =       mean inorganic carbon in the control flasks in mg

Vf=                                                                    volume of the liquid phase in the respective glass in litre

 

Flask

Measured inorganic carbon content

Volume liquid phase

Inorganic carbon in flask (abs.)

Net inorganic carbon content liquid phase

Designation

mg/L

L

mg

mg

Control 1

25.83

0.4912

12.69

 

Control 2

23.52

0.4879

11.48

 

Control 3

23.41

0.4967

11.63

 

Control mean

24.68

 

11.93

 

Pos. Control 1

29.73

0.4926

14.64

2.71

Pos. Control 2

26.25

0.5026

13.19

1.26

Pos. Control 3

26.32

0.507

13.34

1.41

Pos. Control Mean

27.43

 

 

1.80

Test 1

59.53

0.5167

30.76

18.83

Test 2

47.38

0.5087

24.10

12.17

Test 3

59.35

0.5134

30.47

18.54

Test Mean

55.42

 

 

16.51

Toxicity control 1

41.62

0.4997

20.80

8.87

Toxicity control 2

53.95

0.4864

26.24

14.31

Toxicity control 3

63.57

0.5004

31.81

19.88

Toxicity control Mean

53.05

 

 

14.35

 

Elimination carbon in mg

The total eliminated carbon is calculated by addition of the two values.

mt= mh+ mi

with mt= carbon which was transformed to CH4or CO2

 

Flask

Carbon content gas phase

Carbon content liquid phase

Total eliminated carbon

Designation

mg

mg

mg

Pos. Control 1

1.2

2.71

3.9

Pos. Control 2

0.8

1.26

2.0

Pos. Control 3

0.7

1.41

2.1

Pos. Control Mean

0.9

1.80

2.71

Test 1

37.1

18.83

55.92

Test 2

42.1

12.17

54.31

Test 3

38.3

18.54

56.82

Test Mean

39.2

16.51

55.68

Toxicity control 1

40.2

8.87

49.06

Toxicity control 2

46.9

14.31

61.22

Toxicity control 3

52.5

19.88

72.41

Toxicity control Mean

46.5

14.35

60.90

 

Degree of elimination

The degradation is calculated using the following equation:

 

Di = mt x 100 / mz

with

Dt = grade of elimination in %

mt = carbon which was transformed to CH4 or CO2 in mg

mz = organic carbon which was originally added to the test flasks in mg

 

Flask

Total eliminated carbon

Added C in resp. flask

Degradation

Designation

mg

mg

%

Pos. Control 1

3.9

48.9

8.07

Pos. Control 2

2.0

48.9

4.18

Pos. Control 3

2.1

48.9

4.40

Pos. Control Mean

 

 

5.55

Test 1

55.92

50.3

111.2

Test 2

54.31

50.8

106.9

Test 3

56.82

49.8

114.1

Test Mean

 

 

110.7

Toxicity control 1

49.06

98.5

49.8

Toxicity control 2

61.22

98.8

62.0

Toxicity control 3

72.41

99.3

72.9

Toxicity control Mean

 

 

61.6

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance was readily biodegradable under anaerobic conditions.
Executive summary:

The anaerobic biodegradation of the test substance was determined in a test according to OECD guideline 311 (2006) and EN ISO 11734 (1998). Biodegradation was monitored by continuous measurement of the gas developed until the end of the test. Anaerobic sludge was used as inoculum.

After 61 days a degradation of 110.7 % was observed. The degradation exceeding 100 % might be explained by the following: In the test flasks bacterial growth was enhanced by the well biodegradable test substance. After digestion of the test substance the biomass started self-digestion. Self-digestion of the previously increased biomass in the test flasks might very well be higher than the control flasks.

Except for the degradation of the positive control, the validity criteria were met. As the test item was readily and completely biodegradable under test conditions, this deviation from the guideline can be stated as uncritical.

Therefore, the test item is stated to be biodegradable under anaerobic conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please see the attached document “Sophorolipid_Justification for read-across_Ecotox_2015-04-151638581287878619378”.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances, both Sophorolipids have similar environmental fate and pathways, ecotoxicological and toxicological properties based on the underlying identical generic structure. There are only differences in the ratio of the different structures (differentiation between acid and lactone form).
Reason / purpose:
read-across source
Duration of test (contact time):
28 d
Initial conc.:
99.78 mg/L
Based on:
act. ingr.
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
0 d
Parameter:
% degradation (O2 consumption)
Value:
56
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
97
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
> 99
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
> 99
Sampling time:
28 d
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Sufficiently documented guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Inoculum obtained from Chemicals Evaluation and Research Institute
Duration of test (contact time):
28 d
Initial conc.:
99.78 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
- Additional substrate:
- Test temperature: 25 +/- 1 °C

TEST SYSTEM
- Measuring equipment: Closed system oxygen demand measuring device (Coulometer OM3100A (Okura Electric Co., Ltd.
- Test solution system volume: 300 mL
- Amount of test substance added to test solution: 51 mg

MEASUREMENTS
- BOD values measured every sethenth day after day 0

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: no
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
0 d
Parameter:
% degradation (O2 consumption)
Value:
56
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
97
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
> 99
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
> 99
Sampling time:
28 d
Details on results:
The degradation rate was calculated by the following formula:
Degradation = 100 x BOD/ [(TOD x added amount x available concentration / 100) / 0.3)]
Results with reference substance:
Degradation rate after 28 days: 69 %

Δ BOD values*

 

Δ BOD (mg/L)

Day 0

Day 7

Day 14

Day 21

Day 28

Aniline degradation

0

79

201

204

205

Sample degradation

0

85

145

173

177

 *: Δ BOD = (BOD value under each condition) - (BOD value in basic respiration condition)

The degradation rates calculated from the Δ BOD, TOD and ThOD values measured under each condition are shown below:

 

Degradation rate %

Day 0

Day 7

Day 14

Day 21

Day 28

Aniline degradation

0

27

67

68

69

Sample degradation

0

56

97

> 99

> 99

The degradation rate of aniline degradation condition was calculated by the following formula:

Degradation = 100 x Δ BOD / (ThOD x added amount / 0.3)

The degradation rate of the test substance was calculated by the following formula:

Degradation = 100 x Δ BOD / [(ThOD x added amount x available concentration / 100) / 0.3]

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the test the test substance was readily biodegradable.
Executive summary:

The biodegradation of the test substance was determined in an OECD 301 C test (modified MITI test). The degradation was measured by means of oxygen uptake. After 28 days more than 99 % of the test substance was degraded, meeting the 10 day window requirement. Aniline was used as a reference substance with a degradation rate of 69 % after 28 days.

Description of key information

The biodegradation of the substance was investigated in tests under aerobic and under anaerobic conditions. Two studies on the target substance and a supporting read-across study all proved that the substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The biodegradability of Sophorolipids was investigated in three guideline studies.

a. OECD guideline 301F

The biodegradation of the test substance was investigated over a 28 day period in a manometric Respirometry Test according to OECD Guideline 301 F (1992). The test medium was inoculated with microorganisms from a digester of a sewage treatment plant mainly fed with municipal wastewater.

The rate of degradation was monitored by measuring the quantity of oxygen required to maintain a constant gas volume in the respirometer flasks over 28 days.

Sodium benzoate was used as a degradable reference substance at a concentration of 100 mg/L, along with a toxicity control at 100 mg/L test substance and 100 mg/L sodium benzoate. The biodegradation of the test substance was found to be at mean 69 % with a standard deviation of 0.8 % for a concentration of 100 mg test substance/L and at mean 85 % with a standard deviation of 5.4 % for a concentration of 60 mg test item/L. For a concentration of 100 mg test substance/L biodegradation within the 10-day-window was found to be 61 %.

For a concentration of 60 mg test substance/L biodegradation within the 10-day-window was found to be 75 %.

The degradation of of the reference substance sodium benzoate had reached 83 % within the first 14 days.

According to the guideline, the test item is considered as being readily biodegradable under the test conditions.

b. OECD guideline 311

The anaerobic biodegradation of the test substance was determined in a test according to OECD guideline 311 (2006) and EN ISO 11734 (1998). Biodegradation was monitored by continuous measurement of the gas developed until the end of the test. Anaerobic sludge was used as inoculum.

After 61 days a degradation of 110.7 % was observed. The degradation exceeding 100 % might be explained by the following: In the test flasks bacterial growth was enhanced by the well biodegradable test substance. After digestion of the test substance the biomass started self-digestion. Self-digestion of the previously increased biomass in the test flasks might very well be higher than the control flasks.

Except for the degradation of the positive control, the validity criteria were met. As the test item was readily and completely biodegradable under test conditions, this deviation from the guideline can be stated as uncritical.

Therefore, the test item is stated to be biodegradable under anaerobic conditions.

c. Read-across study, OECD 301C test

The biodegradation of the test substance was determined in an OECD 301C test (modified MITI test). The degradation was measured by means of oxygen uptake. After 28 days more than 99 % of the test substance was degraded, meeting the 10 day window requirement. Under the conditions of the test the test substance was readily biodegradable.

Discussion

The test item Sophorolipids was readily biodegradable in both an OECD guideline 301F and an OECD guideline 311 test.

A supporting study based on a read-across approach is available which gave comparable results.