Reaction mass of L-Glutamic acid, N-coco acyl derivs., monosodium salts and sodium hydrogen N-(1-oxooctadecyl)-L-glutamate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2016 to 03 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
-Before the preparation of the respective test solutions with the test material an aqueous stock solution of the test material with a concentration of 28 mg/L was prepared and diluted accordingly. During the preparation of the test solutions they were mixed by mechanical stirring and using ultrasonic bath (5 min.) to ensure a good dispersion. The test material concentration in the test solutions was 2.8 mg/L.
- The chosen test material concentration was based on the measured chemical oxygen demand (COD): 2.12 ± 0.015 mg O2/mg test material and on the performed 14-d preliminary test.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Secondary effluent, microorganisms from a domestic waste water treatment plant in Veszprém, Hungary.
- Storage conditions: The secondary effluent used for this study was allowed to settle for half an hour, then the decanted effluent was aerated until use (not later than an hour).
- Concentration of sludge: 0.5 mL/L

Duration of test (contact time):

28 d

Initial conc.:

2.8 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Adequate amount of the stock solutions a) - d) were combined and filled up with deionised water to the appropriate final volume (ratio of composition referring to 1 mL of the stock solutions a) - d) filled up with deionised water to a final volume of 1000 mL according to the guideline). The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was 9.0 mg/L at about 22 °C.
Stock solutions: In deionised water analytical grade salts were added to prepare the following stock solutions:
a) Solution: KH2PO4 2.125 g, K2HPO4 5.4375 g, Na2HPO4 x 12H2O 16.795 g, NH4Cl 0.125 g and deionised water ad 250 mL.
b) Solution: MgSO4 x 7 H2O 5.625 g and deionised water ad 250 mL.
c) Solution: CaCl2 x 2 H2O 9.10 g and deionised water ad 250 mL.
d) Solution: FeCl3 x 6 H2O 0.25 g and deionised water ad 1000 mL.
- Test temperature: 22 ± 2 °C. The test flasks were placed into an incubator and kept at 21.7 – 23.3 °C. The temperature was measured on weekdays during the experiment.
- pH: The pH value of the test water was checked prior to the start of the experiment and was 7.22 at the start of the test.
- pH adjusted: No
- Aeration of dilution water: The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature.
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: BOD bottles (300 mL) with special neck and glass stoppers. Each test flask was uniquely identified with study code, test group, days of measurement and replicate number.
- Number of culture flasks/concentration: 10
- Measuring equipment: The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode.
- Test performed in closed vessels due to significant volatility of test substance: Yes; tightly closed with glass stoppers.

- Other: BOD flasks were cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then the test solutions were filled into the bottles bubble-free until the bottles were completely filled.
The components were applied in the amounts/volumes in the test flasks:
-Test item flasks 1a and 1b: Based on the measured chemical oxygen demand (COD) of 2.12 mg O2/mg test item, test item stock solution (28 mg/L) (corresponding to 9.52 mg of the test material) was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 2.8 mg/L test item, with a COD of about 5.936 mg O2/L).
-Procedure control flasks 2a and 2b: Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution (360 mg/L) corresponding to 12.240 mg of Sodium benzoate was mixed into 3.40 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L).
-Inoculum control flasks 3a and 3b: Only filtered inoculum was added to 3.40 litres of aqueous test medium.
- Toxicity Control flasks 4a and 4b: Test item stock solution (28 mg/L) (340 mL) and reference item stock solution (360 mg/L) (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 2.8 mg/L test item (COD of 5.936 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L).

SAMPLING
- Sampling frequency: Days 0, 7, 14, 21 and 28.
- The COD (chemical oxygen demand) of the test material was determined in the analytical department of the test facility using Lovibond® COD Measuring System.
- Because of nitrogen content of the test material, samples for nitrate and nitrite analysis were taken from all vessels (of test material, inoculum control and toxicity control group) and the total oxidised nitrogen (nitrate and nitrite) concentrations were determined after each oxygen measurement.
- Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28. The oxygen concentration of the test water was 9.0 mg/L at the start of the test.
- Temperature was measured continuously and registered mostly on each working day, but at least each day of oxygen consumption measurement during the experiment.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 bottles containing only inoculum (inoculum control).
- Toxicity control: 10 bottles containing the test item, reference item and inoculum (toxicity control).
- Procedure control: 10 bottles containing the reference item and inoculum (procedure control).

STATISTICAL METHODS:
-Calculation of BOD: The BOD (mg O2 per mg test material) expected after each period was calculated as follows:

(mg O2/L of T.m and/or R.m – mg O2/L of i.control) / mg T.m and/or R.m/L in flask

where:
T.m = test material
R.m = reference material
i.control = inoculum control

-Calculation of Biodegradation %: The percentage biodegradation of the test material and of the reference material was calculated as follows:

[BOD(mg O2/mg T.m or R.i) / [COD(mg O2/mg T.m) or ThODNH4(mg O2/mg R.m)]] x 100

where:
T.m = test material
R.m = reference material
i.control = inoculum control

Reference substance:

benzoic acid, sodium salt

Test performance:

The study met all of the validity criteria.

Key result

Parameter:

% degradation (O2 consumption)

Value:

75.8

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Value:

62.3

Sampling time:

14 d

Details on results:

-Under the test conditions the percentage biodegradation of the test material reached a mean of 75.8 % after 28 days based on the measured COD of the test material. According to the test guidelines the pass level for ready biodegradability, 60 % of ThOD/COD and this level has to be reached in a 10-day window within the 28-day period of the test. Taking into account that the test material was sufficiently degraded to a mean of 62.3 % after 14 days and according to the test guideline the value obtained in a 14-day window is acceptable in the used method, therefore the test material can be considered to be ready biodegradable.

-In the toxicity control containing both, the test material and the reference material Sodium benzoate, a mean of 53.2 % biodegradation was noted within 14 days and 56.5% biodegradation after 28 days of incubation.
- According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 2.8 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.
- The test material was not considered to have a significant inhibitory effect on the secondary effluent microorganisms.

Results with reference substance:

The reference material Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration [mg/L]	Flask No.	mg O2/L After n Days of Exposure
			0	7	14	21	28
Test Material	2.8	1a	8.8	4.9	4.5	3.8	3.5
		1b	8.9	4.7	4.5	4.0	3.6
		Mean	8.85	4.80	4.50	3.90	3.55
Reference Item	3.6	2a	9.0	4.4	3.7	3.3	2.8
		2b	9.0	4.6	3.8	3.3	3.0
		Mean	9.00	4.50	3.75	3.30	2.90
Inoculum Control	-	3a	9.0	8.6	8.4	8.3	8.2
		3b	9.1	8.6	8.4	8.4	8.3
		Mean	9.05	8.60	8.40	8.35	8.25
Toxicity Control	Test Material: 2.8 Reference Item: 3.6	4a	8.9	2.5	1.8	1.4	1.2
		4b	8.8	2.4	1.7	1.3	1.2
		Mean	8.85	2.45	1.75	1.35	1.20

 

Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration [mg/L]	Flask No.	mg O2/L After n Days of Exposure
			7	14	21	28
Test Material	2.8	1a	3.45	3.65	4.30	4.50
		1b	3.75	3.75	4.20	4.50
Reference Item	3.6	2a	4.15	4.65	5.00	5.40
		2b	3.95	4.65	5.00	5.20
Toxicity Control	Test Material: 2.8 Reference Item: 3.6	4a	5.95	6.45	6.80	6.90
		4b	5.95	6.45	6.80	6.80

oxygen depletion: (mt0 - mtx) - (mb0 - mbx), where:

mt0: oxygen concentration (mg/L) of test group on day 0 (1a, 2a, 4a and 1b, 2b, 4b from Table Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days)

mtx: oxygen concentration (mg/L) of test group on day x (1a, 2a, 4a and 1b, 2b, 4b from Table Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days)

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days)

 

Total Oxidised Nitrogen at concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration [mg/L]	Flask No.	mg Total Oxidized N/L After n Days of Exposure
			0	7	14	21	28
Test Material	2.8	1a	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
		1b	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Inoculum Control	-	3a	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
		3b	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Toxicity Control	Test Material: 2.8 Reference Item: 3.6	4a	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
		4b	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ

LOQ: 4.43 mg NO3/mL; LOQ: 0.03 mg NO2/mL; (< LOQ: below the quantification level)

Equipment: Lovibond Maxidirect Photometer using Nitrite LR tablet reagent and Vario NitraX Reagent Set

 

BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration [mg/L]	Flask No.	BOD After n Days of Exposure
			7	14	21	28
Test Material	2.8	1a	1.23	1.30	1.54	1.61
		1b	1.34	1.34	1.50	1.61
Reference Item	3.6	2a	1.15	1.29	1.39	1.50
		2b	1.10	1.26	1.39	1.44
Toxicity Control	Test Material: 2.8 Reference Item: 3.6	4a	0.93	1.01	1.06	1.08
		4b	0.93	1.01	1.06	1.06

BOD = (mg O2/L of T.i. and/or R.i.- mg O2/L of i.control) / (mg T.i. and/or R.i./L in flask) = mg O2/mg T.i and/or R.i.

where:

T.i. = test material

R.i. = reference item

i.control = inoculum control

 

Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration [mg/L]	Flask No.	Percent of Biodegradation After n Days of Exposure
			7	14	21	28
Test Material	2.8	1a	58.1	61.5	72.4	75.8
		1b	63.2	63.2	70.8	75.8
		Mean	60.6	62.3	71.6	75.8
Reference Item	3.6	2a	69.2	77.5	83.3	90.0
		2b	65.8	75.8	83.3	86.7
		Mean	67.5	76.7	83.3	88.3
Toxicity Control	Test Material: 2.8 Reference Item: 3.6	4a	49.1	53.2	56.1	56.9
		4b	49.1	53.2	56.1	56.1
		Mean	49.1	53.2	56.1	56.5

Biodegradation % = ((BOD (mg O /mg T.i. or R.i.)) / (COD (mg O /mg T.i.) or ThOD (mg O /mg R.i.))) x 100

where:

T.i. = test material

R.i. = reference item

i.control = inoculum control

COD of test material = 2.12 ± 0.015 mg O2/mg test material

ThODNH4 of reference item = 1.67 mg O2/mg reference item

The biodegradation in the toxicity control was calculated according to the following formula:

((BOD (mg O /mg T.i. and R.i.)) / ([COD (mg O /mg T.i.) ThOD (mg O /mg R.i.)] x 0.5)) x 100

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of this study the test material was considered to be readily biodegradable.

Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301D, EU Method C.4-E and OPPTS 835.3110 under GLP conditions in a Closed Bottle Test over a 28 day period.

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference material Sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the test material reached a mean of 75.8 % after 28 days based on the measured COD of the test material. According to the test guidelines the pass level for ready biodegradability, 60 % of ThOD/COD and this level has to be reached in a 10-day window within the 28-day period of the test. Taking into account that the test material was sufficiently degraded to a mean of 62.3 % after 14 days and according to the test guideline the value obtained in a 14-day window is acceptable in the used method, therefore the test material can be considered to be ready biodegradable.

The reference material Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test material and the reference material Sodium benzoate, a mean of 53.2 % biodegradation was noted within 14 days and 56.5% biodegradation after 28 days of incubation.

According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 2.8 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

The study met the validity criteria.

The test material was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

Under the conditions of this study the test material was considered to be readily biodegradable.

Description of key information

Under the conditions of this study the test material was considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301D, EU Method C.4-E and OPPTS 835.3110 under GLP conditions in a Closed Bottle Test over a 28 day period. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301D, EU Method C.4-E and OPPTS 835.3110 under GLP conditions in a Closed Bottle Test over a 28 day period.

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference material Sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the test material reached a mean of 75.8 % after 28 days based on the measured COD of the test material. According to the test guidelines the pass level for ready biodegradability, 60 % of ThOD/COD and this level has to be reached in a 10-day window within the 28-day period of the test. Taking into account that the test material was sufficiently degraded to a mean of 62.3 % after 14 days and according to the test guideline the value obtained in a 14-day window is acceptable in the used method, therefore the test material can be considered to be ready biodegradable.

The reference material Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test material and the reference material Sodium benzoate, a mean of 53.2 % biodegradation was noted within 14 days and 56.5% biodegradation after 28 days of incubation.

According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 2.8 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

The study met the validity criteria.

The test material was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

Under the conditions of this study the test material was considered to be readily biodegradable.