N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD Guideline and GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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 resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2 - 4 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 6 days.
10 mL/L were used to initiate inoculation.
- Initial cell/biomass concentration: 1.0 * 10E9 CFU/L, corresponding to 1.0 * 10E7 CFU/L in the test vessel.

Duration of test (contact time):

28 d

Initial conc.:

15 mg/L

Based on:

test mat.

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 (20.0 - 22.0 °C, short term decrease to 19.5°C (<1 day) and increase to 23°C (<1 day))
- pH adjusted: no
- Continuous darkness: no, low light conditions (brown glass bottles)



TEST SYSTEM
- Culturing apparatus: 5000 mL brown glass flasks
- Number of culture flasks/concentration: 1 for the reference item, 1 for toxicity control (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. Test and reference item were weighed out and were transferred into the incubation vessels with bidistilled water. 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:

benzoic acid, sodium salt

Remarks:

20 mg/L

Preliminary study:

Not performed

Key result

Parameter:

% degradation (CO2 evolution)

Value:

10

Sampling time:

28 d

Details on results:

The adaptation phase of the functional control changed after 1 day into the degradation phase (degradation > 10 %). The course of the degradation was rapid and the functional control reached the pass level of 60 % after 6 days and a biodegradation of 102 % after 28 days. The validity criterion degradation > 60 % after 14 days is fulfilled.

Results with reference substance:

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 6 days and a biodegradation of 102 % after 28 days.
In the toxicity control containing both test and reference item a biodegradation of 37 % was determined within 14 days and it came to 40 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.
In the toxicity control a biodegradation of 37 % was determined within 14 days and it came to
40 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The biodegradation of the test item is shown graphically in comparison to the readily degradable functional control and the toxicity control. The 1st item replicate reached the 10 % level (beginning of biodegradation) after 28 days and came to a biodegradation of 11 % after 28 days. The 2nd item replicate did not reach the 10 % level.
The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.

CO₂-Production and Biodegradation after 28 Days

CO2-Production	Functional	Test Item		Toxicity Control
after 28 d	Control	15 mg/L		15 mg/L Test Item +
	20 mg/L	No.1	No. 2	20 mg/L Reference Item
Net       [mg/3 L]	130.9	13.6	9.5	99.1
[mg/L]	43.6	4.5	3.2	33.0
Theor.   [mg/3 L]	127.8	121.05	121.05	248.85
[mg/L]	42.6	40.4	40.4	82.95
Degradation [%] after 28 d	102	11	8	40

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 15 mg/L Test Item + 20 mg/L Reference Item
		[mg CO2/3 L]	[mg CO2/3 L]		Degr.	[mg CO2/3 L]		Degr.
		mv	Gross	Net Sum	[%]	Gross	Net Sum	[%]
1	2011-09-23	6.0	26.7	20.7	16	22.8	16.8	7
4	2011-09-26	14.8	50.6	56.5	44	51.7	53.7	22
6	2011-09-28	12.8	39.3	83.0	65	34.8	75.7	30
8	2011-09-30	12.1	21.6	92.5	72	19.5	83.1	33
12	2011-10-04	19.0	25.3	98.8	77	25.4	89.5	36
14	2011-10-06	13.4	20.2	105.6	83	15.2	91.3	37
18	2011-10-10	13.1	26.1	118.6	93	20.9	99.1	40
21	2011-10-13	23.2	30.3	125.7	98	18.9	99.1	40
25	2011-10-17	41.1	43.2	127.8	100	18.9	99.1	40
28	2011-10-20	33.1	33.4	128.1	100	17.2	99.1	40
29*	2011-10-21	28.3	31.1	130.9	102	20.2	99.1	40

                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 15 mg/L
		Control	Replicate 1			Replicate 2
		[mg CO2/3 L]	[mg CO2/3 L]		Degr.	[mg CO2/3 L]		Degr.
		mv	Gross	Net Sum	[%]	Gross	Net Sum	[%]
1	2011-09-23	6.0	5.8	0.0	0	5.8	0.0	0
4	2011-09-26	14.8	13.6	0.0	0	13.6	0.0	0
6	2011-09-28	12.8	12.3	0.0	0	11.8	0.0	0
8	2011-09-30	12.1	8.3	0.0	0	12.1	0.0	0
12	2011-10-04	19.0	19.4	0.4	0	23.9	4.9	4
14	2011-10-06	13.4	8.7	0.4	0	15.2	6.7	6
18	2011-10-10	13.1	11.9	0.4	0	15.6	9.2	8
21	2011-10-13	23.2	25.9	3.1	3	19.3	9.2	8
25	2011-10-17	41.1	43.9	5.9	5	27.6	9.2	8
28	2011-10-20	33.1	38.1	10.9	9	30.4	9.2	8
29*	2011-10-21	28.3	31.0	13.6	11	22.1	9.5	8

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

Validity criteria fulfilled:

yes

Conclusions:

The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.

Executive summary:

The ready biodegradability of the test item was determined with a non adapted activated sludge over a test period of 28 days in the Modified Sturm Test according to OECD 301 B. The test item was tested at a concentration of 15 mg/L in duplicates, corresponding to a total organic carbon content (TOC) of 11.0 mg C/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 hours. The percentage CO₂ production was calculated in relation to the theoretical CO₂ production (ThCO₂) of the test item. The biodegradation 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 6 days and a biodegradation of 102 % after 28 days.

In the toxicity control containing both test and reference item a biodegradation of 37 % was determined within 14 days and it came to 40 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The biodegradation of the test item is shown graphically in Figure 1 in comparison to the readily degradable functional control and the toxicity control. The 1^st item replicate reached the 10 % level (beginning of biodegradation) after 28 days with a biodegradation of 11 %. The 2^nd item replicate did not reach the 10 % level. (8%)

Biodegradation of the Test Item
                   in Comparison to the Functional Control and Toxicity Control

	Biodegradation [%]
	Study Day [d]
	6	14	21	28
Test Item, 1^st Replicate 15 mg/L	0	0	3	11
Test Item, 2^nd Replicate 15 mg/L	0	6	8	8
Functional Control 20 mg/L	65	83	98	102
Toxicity Control 15 mg/L Test Item + 20 mg/L Reference Item	30	37	40	40

                       

Description of key information

The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.