Sophorolipids: fermentation products of glucose and fatty acids, C18 (unsaturated), glycerol esters with yeast Candida Bombicola, partially hydrolysed

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

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

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

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. QA statement)

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.

Reference 2

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

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

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. QA statement)

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:

M_h= 0.468 x Δp_kum x V_h

with

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

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

               p_T(1) – p_BI(1) + p_T(2) + …p_T(n) – p_BI(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:

m_i = IC(Test) x V_f(Test) – Σ³_i-1[IC(Control)_I x V_f(Control)_i]/3

with

m_i =                                                                     inorganic carbon content in the liquid phase in mg

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

Σ³_i-1[IC(Control)_I x V_f(Control)_i]/3 =       mean inorganic carbon in the control flasks in mg

V_f=                                                                    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.

m_t= m_h+ m_i

with m_t= 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

D_t = grade of elimination in %

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

m_z = 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.

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 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.