N,N'-ethylenedi(diacetamide)

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-03-26 to 2009-04-24

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethylenedi(diacetamide)

EC Number:

212-811-9

EC Name:

N,N'-ethylenedi(diacetamide)

Cas Number:

871-78-3

Molecular formula:

C6H12N2O2

IUPAC Name:

N-(2-acetamidoethyl)acetamide

Constituent 2

Reference substance name:

Diacetylethylenediamine (DAED)

IUPAC Name:

Diacetylethylenediamine (DAED)

Details on test material:

- Name of test material (as cited in study report): N,N'-Diacetylethylenediamine (DAED)
- Physical state: Solid, powder
- Analytical purity: 99.9 %
- Lot/batch No.: 14-016
- Expiration date of the lot/batch: 2010-05-21
- Storage condition of test material: Room temperature, protected from moisture and light

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal sewage treatment plant, D-31137 Hildesheim, Germany
- Pretreatment:
- Concentration of sludge: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for four hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air for 5 days- 1 mL/L were used to initiate inoculation.
- Initial cell/biomass concentration: 1.3 x E9 CFU/L

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

11.6 other: mg C/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
- Test temperature: 22 +/- 2°C
- pH adjusted: no
- Continuous darkness: no, low light conditions



TEST SYSTEM
- Culturing apparatus: 5000 mL brown glass flasks
- Number of culture flasks/concentration: 1 for the reference item, 1 for the test and reference item, 2 for the control, 2 for the test item
- Method used to create aerobic conditions: Aeration with 30 - 100 mL/min
- Measuring equipment: Visual check of aeration twice per day
- Details of trap for CO2 and volatile organics if used: CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.
- Other: Application: The necessary amounts of bidistilled water, mineral salts medium and inoculum were placed in each of the incubation vessels. The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels. The test item and reference item were weighed out. A defined volume of bidistilled water was added to the test item and it was treated with ultrasound. The dispersions and the reference item were transferred into the incubation vessels. The vessels were made up to 3 L with bidistilled water and connected to the system for the production of CO2 free air.


SAMPLING
- Sampling frequency: Backtitration of the residual Ba(OH)2 with 0.05 N HCL was carried out three times a week during the first ten days and thereafter twice weekly.
- Sampling method: For each titration the first gas wash bottle was removed and a new bottle was connected to the last one.



CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentration



STATISTICAL METHODS:
The theoretical production of carbon dioxide (ThCO2) of the test item and functional control is calculated by the carbon content and the molecular formula, respectively.
The produced CO2 was calculated by: 1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2
The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the inoculum controls.
The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

20 mg/L

Results and discussion

Preliminary study:

Not performed

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

99

Sampling time:

28 d

Details on results:

The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements.
The test item reached the 10 % level (beginning of biodegradation) after 2 days and the pass level of 60 % after 7 days. The mean biodegradation rate after 28 days was 99 %.

The test item must be regarded as readily biodegradable in the 10-d-window and after 28 days.

BOD5 / COD results

Results with reference substance:

The adaptation phase of the functional control changed after 2 days into the degradation phase (degradation >/= 10 %). The course of the degradation was rapid and the functional control reached the pass level of 60 % after 7 days and came to a maximum of 82 % after 28 days. The validity criterion degradation >/= 60 % after 14 d is fulfilled.
In the toxicity control a biodegradation rate of 78 % was determined within 14 days and it came to 85 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Any other information on results incl. tables

CO₂-Production and Biodegradation after 28 Days

CO2-Production	Functional	Test Item		Toxicity Control
after 28 d	Control	25 mg/L		25 mg/L Test Item +
	20 mg/L	No.1	No. 2	20 mg/L Reference Item
Net       [mg/3 L]	105.1	142.1	125.8	216.5
[mg/L]	35.0	47.4	41.9	72.2
Theor.   [mg/3 L]	127.8	128.3	128.3	256.1
[mg/L]	42.6	42.8	42.8	85.4
Degradation [%] after 28 d	82	100	98	85

CO₂-Production and Biodegradation in the Inoculum Control, Functional Control
                   and Toxicity Control Samples

Study Day	Date	Inoculum Control	Functional Control 20 mg/L			Toxicity Control 25 mg/L Test Item + 20 mg/L Reference Item
		[mg CO2/3L]	[mg CO2/3L]		Degr.	[mg CO2/3L]		Degr.
		mv	Gross	Net Sum	[%]	Gross	Net Sum	[%]
1	27.03.09	8.3	12.7	4.4	3	14.1	5.8	2
4	30.03.09	15.3	54.5	43.6	34	54.5	45.0	18
6	01.04.09	11.5	42.8	74.9	59	54.5	88.0	34
8	03.04.09	9.7	20.9	86.1	67	54.2	132.5	52
11	06.04.09	8.9	15.6	92.8	73	54.3	177.9	69
14	09.04.09	12.3	19.1	99.6	78	34.4	200.0	78
19	14.04.09	14.3	17.5	102.8	80	23.3	209.0	82
21	16.04.09	10.7	11.4	103.5	81	15.5	213.8	83
25	20.04.09	16.5	15.2	103.5	81	17.6	214.9	84
28	23.04.09	14.3	14.1	103.5	81	15.6	216.2	84
29*	24.04.09	11.2	12.8	105.1	82	11.5	216.5	85

                   Degr. = degradation              mv = mean value
                   *) results of last two gas wash bottles

CO₂-Production and Biodegradation in the Inoculum Control and Test Item Samples

Study Day	Date	Inoculum	Test Item 25 mg/L
		Control	Replicate 1			Replicate 2
		[mg CO2/3L]	[mg CO2/3L]		Degr.	[mg CO2/3L]		Degr.
		mv	Gross	Net Sum	[%]	Gross	Net Sum	[%]
1	27.03.09	8.3	7.5	0.0	0	6.2	0.0	0
4	30.03.09	15.3	51.6	36.3	28	52.4	37.1	29
6	01.04.09	11.5	43.6	68.4	53	40.2	65.8	51
8	03.04.09	9.7	30.6	89.3	70	28.5	84.6	66
11	06.04.09	8.9	26.2	106.6	83	24.5	100.2	78
14	09.04.09	12.3	27.5	121.8	95	22.8	110.7	86
19	14.04.09	14.3	30.5	138.0	100	23.9	120.3	94
21	16.04.09	10.7	12.5	139.8	100	10.3	120.3	94
25	20.04.09	16.5	17.3	140.6	100	21.8	125.6	98
28	23.04.09	14.3	15.8	142.1	100	14.5	125.8	98
29*	24.04.09	11.2	10.1	142.1	100	9.1	125.8	98

                                   Degr. = degradation         mv = mean value
                          *) results of last two gas wash bottles

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item must be regarded as readily biodegradable in the 10-d-window and after 28 days.

Executive summary:

The ready biodegradability of the test item N,N’-Diacetylethylenediamine (DAED) (batch no.:   14-016) was determined with a non adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted from 2009-03-26to 2009-04-24according to OECD 301 B at Dr.U.Noack-Laboratorien. The test item was tested at a concentration of  25 mg/L in duplicates, corresponding to a carbon content (TOC) of 11.6 mgC/L in the test vessels. The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO₂ produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29, after residual CO₂ had been purged from the test solutions over a period of 24 h. The percentage CO₂production was calculated in relation to the theoretical CO₂production (ThCO₂) of the test item. The biodegradation rate was calculated for each titration time.

To check the activity of the test system, sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % after 7 days and came to a maximum of 82 % after 28 days.

In the toxicity control containing both test and reference item a biodegradation rate of 78 % was determined within 14 days and it came to 85 % after 28 days.The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The test item reached the 10 % level (beginning of biodegradation) after 2 days and the pass level of 60 % after 7 days. The mean biodegradation rate after 28 days was 99 %.

The test item must be regarded as readily biodegradable in the 10-d-window and after 28 days.

Biodegradation of the Test Item N,N’-Diacetylethylenediamine (DAED) in
                   Comparison to the Functional Control and Toxicity Control

	Biodegradation [%]
	Study Day [d]
	6	14	21	28
Test Item, 1^st Replicate 25 mg/L	53	95	100	100
Test Item, 2^nd Replicate 25 mg/L	51	86	94	98
Functional Control 20 mg/L	59	78	81	82
Toxicity Control 25 mg/L Test Item + 20 mg/L Reference Item	34	78	83	85