Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 October - 30 January 2017

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:

2006

GLP compliance:

yes

Inoculum or test system:

digested sludge

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): municipal wastewater treatment plant Breisgauer Bucht
- Storage conditions: The inoculum was kept before use for 3 days under test conditions in order
to reduce the blank values.
- Preparation of inoculum for exposure: It was then washed three times by centrifugation at 3000 g for 10 min and decanting the supernatant, resuspending the sediment each time in anaerobic mineral medium prepared (without S1 and S5).
- Pretreatment: To determine the amount of total dry solids three samples of digesting sludge were filtered and then dried at 105°C until the dry solids weight
remained constant. The dry solid of the used sludge was determined as being 29.7 g/L.
- Concentration of sludge:
- Initial cell/biomass concentration: The measured IC after centrifugation and resuspension in mineral medium (twice) was 62.07 mg/L. The calculated IC in the test vessel was 6.26 mg/L.
- Water filtered: yes
- Type and size of filter used, if any: folded paper filters, ∅ 12.5 cm, Machery Nagel

Duration of test (contact time):

60 d

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

107.5

Sampling time:

41 d

Remarks on result:

other: Readily biodegradable

Results with reference substance:

The degradation of the reference item started after a lag phase of about 14 days and reached a
mean degradation extent of 90% on day 41 (after considering the IC in the liquor, measurement
of four flasks).
The pH at the end of the test was 6.8 in all reference vessels.

Validity criteria fulfilled:

yes

Remarks:

The test bottles do not show pink coloration after 24 - 48 h; The percentage degradation of the reference item has reached a plateau that represents more than 60% biodegradation; The pH at the end of the test is in the range 7 ± 1

Interpretation of results:

other: The biodegradation at the end of the test was >100%.

Conclusions:

The mean degradation of the test item was 107.5% within 41 days after considering the IC in the
liquor (mean value of four measurements). The single degradation extents ranged from 98% to
119%.
The test item is biodegradable under anaerobic conditions.
The biodegradation at the end of the test was >100%. An incorrect carbon content of the test item
can be excluded as cause, since the determined carbon content was additionally confirmed by
an external analytical laboratory. Therefore the increased biodegradation value probably results
from leaching effects. The test item may have redissolved organic substances adsorbed onto the
sludge, leading to a higher carbon content in the test vessels and an increased gas production.
The pH at the end of the test was 6.7 – 6.8.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-03 to 2014-07-31

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:

2008

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 (e.g. location, sampling depth, contamination history, procedure): Fresh samples of activated sludge from the sewage treatment plant Ruhrverband Klaranlage, Sunthelle 6, 57392 Schmallenberg, Germany, mainly fed with municipal wastewater
-Storage conditions: The samples were not washed with mineral medium after the arrival at the laboratory but kept aerobic until use.
- Concentration of sludge: The concentration used in the test was 29.6 mg dry mass/litre (7.40 mg dry mass/250 mL).

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

act. ingr.

Remarks:

173 mg ThOD

Initial conc.:

32 mg/L

Based on:

act. ingr.

Remarks:

55 mg ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
(a) KH2PO4 8.50 g/L
K2HPO4 21.74 g/L
Na2HPO4 x 12 H2O 67.13 g/L
NH4Cl 0.49 g/L
adjusted to 7.4 ± 0.2 with H2SO4 (50 g/L)
(b) CaCl2 x 2 H2O 36.42 g/L
(c) MgSO4 x 7 H2O 22.49 g/L
(d ) FeCl3 x 6 H2O 0.24 g/L.
- Test temperature: 22 ± 1°C
- pH: 7.4 ± 0.2
- pH adjusted: no
- Suspended solids concentration: 7.40 mg dry mass/250 mL
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Measuring equipment: SAPROMAT respirometer (VOITH lnc.)

SAMPLING
- Sampling frequency: Oxygen demand was carried out continuously throughout the test course.
- Sterility check if applicable: An abiotic control containing test item at 100 mg per litre sterilized mineral test medium (25 mg/250 mL) was applied. The assay was sterilized by adding HgCl2.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 vessels inoculum only
- Abiotic sterile control: 2 vessels test item (100 mg/L) and sterilising agent
- Toxicity control: 2 vessels test item (100 mg/L), reference item (100 mg/L) and inoculum

Reference substance:

benzoic acid, sodium salt

Test performance:

A measured volume of inoculated mineral medium, containing a known concentration of test item (usually 100 mg test item/L giving at least 50-100 mg ThOD/L) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature of 22 ± 1°C The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas volume in the respirometer flask. Evolved carbon dioxide is absorbed in a suitable absorbent.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test suspension A

Value:

68

St. dev.:

0

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test suspension B

Value:

95.9

St. dev.:

3.8

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test suspension A

Value:

84.2

St. dev.:

2.5

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test suspension B

Value:

105.8

St. dev.:

0

Sampling time:

28 d

Details on results:

The biodegradation of the test item after 28 days of incubation in the static test was found to be 84% (SD = 2.5%) and 105% (SD = 0.0%) in the assays with 100 mg/L and 32 mg/L, respectively. The biodegradation within the 10-day-window was 63% and 92% in the assays with 100 mg/L and 32 mg/L, respectively. The 10-day-window started at day 1 - 2, independent from test item concentration. The degradation rate >100% in the test assays with 32 mg test item per liter is assumed to be due to cometabolism.
With 1%, 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 62 % 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.

Results with reference substance:

The reference item sodium benzoate was degraded to 81 % within the first 14 days.

Table 1: Degradation / elimination kinetics

Type of suspension	Vessel no.	% degradation at sampling time (days)
		0	t1	t2	t3	t4	t5	t6	t7	t8	t9	t10	t11	t12	t13	t14	t15	t16	t17	t18	t19	t20	t21	t22	t23	t24	t25	t26	t27	t28
Abiotic sterile control	1	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.6	0.6	0.6	0.6
	2	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.6	0.6	0.6
Procedural control	1	0.0	18.0	36.6	45.6	52.2	59.4	65.1	68.5	70.6	71.8	73.0	73.6	74.2	75.4	76.0	76.9	77.2	77.2	77.2	76.9	76.6	77.2	77.2	77.2	76.9	77.2	77.2	77.2	77.2
	2	0.0	25.2	40.8	50.4	69.7	75.7	79.0	81.1	82.0	83.2	84.4	85.0	85.0	85.6	86.2	86.5	86.8	86.8	87.4	87.7	87.4	88.0	88.6	88.6	88.9	89.2	89.2	89.8	89.8
Toxicity control	1	0.0	15.0	21.8	25.9	31.2	35.4	39.3	43.3	47.3	50.8	54.4	57.6	60.3	63.2	65.9	68.1	70.0	72.1	73.8	75.2	76.5	78.0	79.4	80.6	81.6	82.7	84.1	85.0	85.9
	2	0.0	15.3	21.2	24.8	29.8	33.0	36.1	39.2	42.3	44.9	47.3	50.0	52.3	55.3	58.5	61.3	63.8	66.5	68.2	69.6	70.9	72.1	73.2	74.4	75.4	76.5	77.7	78.5	79.4
Test sample A	1	0.0	2.3	11.0	15.0	17.9	22.6	28.7	34.1	40.8	47.8	54.1	57.6	60.5	64.0	68.0	70.0	72.6	74.4	76.1	77.0	77.8	78.4	79.6	80.7	80.5	81.3	81.3	82.5	82.5
	2	0.0	5.8	13.9	17.9	20.8	24.3	30.4	37.6	44.9	51.8	57.0	59.9	62.2	65.1	68.0	71.2	73.8	76.7	79.0	79.9	81.3	81.9	83.1	83.6	83.9	84.8	85.4	85.4	86.0
Test sample B	1	0.0	12.7	28.9	38.0	59.7	70.5	76.9	81.4	85.9	89.5	91.3	85.0	95.0	96.8	98.6	99.5	100.4	102.2	102.2	101.3	102.2	104.0	104.0	104.0	103.1	104.0	105.8	105.8	105.8
	2	0.0	9.0	23.5	28.9	56.1	65.1	73.3	77.8	82.3	84.1	87.7	89.5	89.5	91.3	93.2	92.2	93.2	95.0	96.8	97.7	98.6	100.4	102.2	102.2	101.3	102.2	104.0	105.8	105.8

Validity criteria:

- With 0 - 3% the difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20%.

- The percentage degradation of the reference item has exceeded the pass level of 60% by day 14.

- The oxygen uptake of the inoculum blank is < 60 mg/L in 28 days and the pH value was inside the range of 6.0 - 8.5.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The present study was conducted to determine the biodegradability of Rhamnolipids. The biodegradation was found to be at mean 84% (100 mg test item per liter) and 106% (32 mg test item per liter) after 28 days, and 63% (100 mg test item/L) and 92% (32 mg test item/L) within the 10 days window. Thus, the test item is considered to be readily biodegradable.

Executive summary:

The biodegradation of Rhamnolipids (100% a.i.) was investigated over a 28-day period in aManometric Respirometry Testaccording to OECD Guideline 301 F (1992) and EU method C.4-D (2008). The test medium was inoculated withactivated sludge from 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. The test item was tested in a concentration of 100 mg/L (ThOD = 173 mg/L) and 32 mg/L (ThOD = 55 mg/L) in duplicates.

In order to check the procedure, sodium benzoate was used as a degradable reference item at a concentration of 100 mg/L, along with a toxicity control at 100 mg/L test item and 100 mg/L sodium benzoate.

The biodegradation was found to be at mean 84% (100 mg test item/L) and 106% (32 mg test item/L) after 28 days, and 63% (100 mg test item/L) and 92% (32 mg test item/L) within the 10 days window.

The degradation of the reference substance sodium benzoate had reached 81% within the first 14 days. The difference of extremes of replicate values of the removal of the test item at the end of the test for 100 mg/L is less than 20%. Therefore, the test can be considered as valid. No inhibitory effects of the test item were observed (more than 25% degradation occurred within 14 days) in the toxicity control.

According to the guideline, Rhamnolipids can be considered as readily biodegradable under the chosen test conditions.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable