2,4,6-trimethylbenzene-1,3-diamine

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:

2016-04-01 to 2016-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

/ Regulation 440/2008/EC C. 4-D

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): Activated sludge from the municipal sewage treatment plant, 31137 Hildesheim, Germany
- Preparation of inoculum for exposure: 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 2 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 and/or stirring until test start. 10 mL/L were used to initiate inoculation.
- Initial cell/biomass concentration: 10e7 - 10e8 CFU/L
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301F
- Additional substrate: No
- Test temperature: 20 - 20.9 °C
- pH:
pH-Values at Test Start and Test End
pH-Value
Start End
Inoculum Control 7.74 1) 7.73
2) 7.72
Functional Control 7.76 7.88
Test Item 7.77 1) 7.77
2) 7.75
Toxicity Control 7.75 8.08

- pH adjusted: no
- Dispersion treatment: Continuous stirring
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Closed bottles with a rubber sleeve with soda lime
- Measuring equipment: The bottles were closed with OxiTOP measuring heads.
The oxygen consumption was determined continuously from the pressure in the incubation vessels by OxiTop measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.

SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Sampling method: Pressure measurements directly from the test vessels


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 concentrations


STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD) of the test item and functional control.
Concerning the OECD-guideline 301 the index of the atoms of the item CcHhClclNnNanaOoPpSs was used for the calculation of the ThOD.
The oxygen depletion of the test and reference item was calculated out of the difference of the test and inoculum control replicates.
The biodegradation of the test and reference item was calculated out of the BOD in relation to the ThOD.

Reference substance:

other: Sodium benzoate

Preliminary study:

No preliminary study

Test performance:

Based on the calculated oxygen demand, the test concentration of 30 mg/L, corresponding to an oxygen demand of 70.2 mg O2/L (95.7 mg O2/L with nitrification) in the vessel, was selected. The test solutions were prepared in measuring flasks and were placed in brown glass bottles as incubation vessels (inoculum control, functional control, test item and toxicity control):
• two for the inoculum control (C1, C2)
• one for the functional control (R1)
• two for the test item (P1, P2)
• one for the toxicity control (T1)
Inoculum control
The inoculated test medium, consisting of the required volumes of mineral medium stock solutions, ultrapure water and 10 mL/L inoculum, were prepared in a measuring flask. 250 mL of this solution were filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask.

Functional control
For the functional control, the reference item was weighed out and transferred into a measuring flask, the required volumes of mineral medium stock solutions, ultrapure water and inoculum, were added. 250 mL of this solution was filled in the brown glass bottle of the functional control, using a 250 mL measuring flask.

Test item
For the test item replicates the test item was weighed out and transferred into a measuring flask, the required volumes of mineral medium stock solutions, ultrapure water and inoculum were added. 250 mL of this solution were filled in the brown glass bottles of the test item rep-licates, using a 250 mL measuring flask.

Toxicity control
For the toxicity control, the test and reference item were weighed out and transferred into a measuring flask, the required volumes of min-eral medium stock solutions, ultrapure water and inoculum, were add-ed. 250 mL of this solution were filled in the brown glass bottle of the toxicity control, using a 250 mL measuring flask, using a 250 mL measuring flask.

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

Details on results:

The mean oxygen depletion in the inoculum control was 7.7 mg O2/L on day 28.

In the toxicity control containing both test and reference item 45% degradation occurred within 14 days. After 28 days the biodegradation came to 46%. The degradation of the reference item was not inhibited by the test item.

Both test item replicates did not reach the 10% level (beginning of biodegradation) until test end. The mean biodegradation on day 28 was 3%.

The validity criteria of the guideline are fulfilled.

Results with reference substance:

In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60% was reached within 3 days. The biodegradation reached a maximum of 100 % degradation on day 13.

Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control, Test Item and Toxicity Control

		BOD [mg O2/L]
Date	[d]	Inoculum Control	Functional Control		Test Item				Toxicity Control Test Item + Reference Item
			R1		P1		P2		T1
		mean	gross	net	gross	net	gross	net	gross	net
2016-04-14	1	1.1	9.9	8.8	2.0	1.0	1.9	0.9	7.8	6.7
2016-04-15	2	1.6	43.0	41.4	4.2	2.6	4.0	2.4	39.9	38.3
2016-04-16	3	2.2	53.8	51.6	4.2	2.0	4.2	2.0	47.6	45.4
2016-04-17	4	2.7	62.9	60.2	5.1	2.4	4.2	1.5	56.1	53.4
2016-04-18	5	3.1	67.2	64.1	5.4	2.3	4.2	1.1	61.0	58.0
2016-04-19	6	4.7	71.4	66.6	7.8	3.1	5.6	0.9	63.6	58.9
2016-04-20	7	5.2	73.0	67.8	8.5	3.3	5.7	0.5	65.4	60.2
2016-04-21	8	5.4	74.8	69.4	8.5	3.1	6.1	0.7	66.5	61.1
2016-04-22	9	5.5	76.0	70.4	8.5	3.0	6.2	0.7	67.8	62.3
2016-04-23	10	5.6	77.3	71.7	8.6	3.0	6.8	1.2	68.6	63.0
2016-04-24	11	5.6	78.7	73.1	9.4	3.8	6.9	1.3	69.1	63.5
2016-04-25	12	5.6	80.0	74.4	9.7	4.1	6.9	1.3	69.8	64.2
2016-04-26	13	5.7	80.7	75.1	9.8	4.1	6.9	1.2	70.2	64.5
2016-04-27	14	5.7	81.7	76.0	9.9	4.2	7.0	1.3	70.5	64.8
2016-04-28	15	6.0	82.6	76.5	9.2	3.2	6.7	0.6	71.0	65.0
2016-04-29	16	4.5	81.7	77.2	7.0	2.5	5.6	1.1	70.5	66.0
2016-04-30	17	4.8	82.7	77.9	7.0	2.2	5.6	0.8	70.8	66.0
2016-05-01	18	5.0	83.0	78.0	7.0	2.0	6.3	1.3	71.3	66.3
2016-05-02	19	5.2	83.1	77.9	7.0	1.8	5.9	0.8	71.8	66.6
2016-05-03	20	4.8	83.1	78.3	7.0	2.2	6.4	1.6	71.9	67.1
2016-05-04	21	6.0	84.7	78.7	9.2	3.2	7.0	1.0	73.0	66.9
2016-05-05	22	5.3	84.4	79.1	7.6	2.2	7.0	1.7	72.8	67.4
2016-05-06	23	5.6	84.5	78.9	8.0	2.4	7.0	1.4	73.3	67.7
2016-05-07	24	5.6	84.5	78.9	7.9	2.3	7.0	1.4	73.3	67.7
2016-05-08	25	5.5	84.5	79.0	8.1	2.6	7.0	1.5	73.3	67.8
2016-05-09	26	5.8	85.0	79.1	8.7	2.9	7.2	1.4	73.4	67.6
2016-05-10	27	7.7	87.4	79.7	11.3	3.6	8.5	0.8	74.1	66.4
2016-05-11	28	7.7	87.4	79.7	11.3	3.6	8.5	0.8	74.3	66.6

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

		Biodegradation [%]
Date	[d]	Functional Control	Test Item		Toxicity Control Test Item + Reference Item
		R1	P1	P2	T1
2016-04-14	1	12	1	1	5
2016-04-15	2	55	4	3	26
2016-04-16	3	69	3	3	31
2016-04-17	4	80	3	2	37
2016-04-18	5	85	3	2	40
2016-04-19	6	89	4	1	40
2016-04-20	7	90	5	1	41
2016-04-21	8	92	4	1	42
2016-04-22	9	94	4	1	43
2016-04-23	10	95	4	2	43
2016-04-24	11	97	5	2	44
2016-04-25	12	99	6	2	44
2016-04-26	13	100	6	2	44
2016-04-27	14	100	6	2	45
2016-04-28	15	100	5	1	45
2016-04-29	16	100	4	2	45
2016-04-30	17	100	3	1	45
2016-05-01	18	100	3	2	46
2016-05-02	19	100	3	1	46
2016-05-03	20	100	3	2	46
2016-05-04	21	100	5	1	46
2016-05-05	22	100	3	2	46
2016-05-06	23	100	3	2	47
2016-05-07	24	100	3	2	47
2016-05-08	25	100	4	2	47
2016-05-09	26	100	4	2	46
2016-05-10	27	100	5	1	46
2016-05-11	28	100	5	1	46

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

After a test period of 28 days, the test item is classified as not readily biodegradable within 28 days.

Executive summary:

The ready biodegradability of the test item was determined with non-adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted according to OECD guideline 301 F and regulation 440/2008/EC C.4-D at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen.

The test item concentration selected as appropriate was 30.0 mg/L, corresponding to a ThOD of 70.2 mg O₂/L per test vessel. concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 7.7 mg O₂/L on day 28. Both test item replicates did not reach the 10% level (beginning of biodegradation) until test end. The mean biodegradation on day 28 was 3%. The test item is classified as not readily biodegradable within 28 days

Description of key information

The ready biodegradability of the test item was determined with non-adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted according to OECD guideline 301 F and regulation 440/2008/EC C.4-D.

The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen.

The test item concentration selected as appropriate was 30.0 mg/L, corresponding to a ThOD of 70.2 mg O₂/L per test vessel. concentration. Both test item replicates did not reach the 10% level (beginning of biodegradation) until test end. The mean biodegradation on day 28 was 3%.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed