N,N'-bis(1,3-dimethylbutylidene)ethylenediamine

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:

2021-07-16 to 2021-08-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 17th July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Chemical Name: N,N’-Bis(1,3-dimethylbutylidene)ethylenediamine
- CAS No.: 25707-70-4
- EC No.: 247-196-6

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 09 July, 2021 (seven days before the test)
- Storage conditions:continuously aerated (2 L/minute) at the test temperature of 20.0 – 20.9 °C
- Storage length: 7 days
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution by shaking and was again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.555 g wet weight), dried and the ratio of wet sludge to dry weight (0.5115 g dry weight) was determined. Based on this ratio, calculated amount of wet sludge (5 g dry weight that was equivalent to 54.3 g wet sludge) was suspended in mineral medium (ad. 1000 mL) to yield a concentration equivalent to about 5 g per litre (on dry weight basis). The prepared activated sludge inoculum was aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.52, just before use the pH was: 6.74. A pH adjustment of activated sludge inoculum was not performed.

Pre-conditioning of Activated Sludge Inoculum: Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium4) for 7 days at the test temperature (the actual temperature: 20.0 – 20.9 °C). Before use the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating of 0.1 mL undiluted sample and different, 10-1, 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates. Plates were incubated at 37 °C for 24 hours. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. During the pre-conditioning the approximately cell count of aerated inoculum was ~ 108 /L; therefore, before the test the inoculum was further diluted 100 000x with mineral medium to reach the necessary ~104 – 106 /L cell concentration. After preparation the sludge was filtered through cotton wool. Preconditioning improved the precision of the test methods by reducing blank values. The inoculum was not pre-adapted to the test chemical.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

ThOD

Remarks:

ThODNH4

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: according to guideline
- Additional substrate: no
- Test temperature: 20.4 - 21.6 °C.
- pH: The pH of the activated sludge inoculum after preparation was 7.52, just before use the pH was: 6.74.
- 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: incubator with thermometer with exclusion of ligh in a temperature controlled (22 ± 2 °C) environment room
- Number of culture flasks/concentration: 10 (+ 2 reserve) bottles containing the test item and inoculum
- Method used to create aerobic conditions: test medium and inoculum aerated
- Measuring equipment: Oxygen and pH meter with appropriate O2 and pH electrode, spectrophotometer (for nitrate and nitrite determination)
- Test performed in open system: no


SAMPLING
- Sampling frequency: Oxygen measurements were performed in all duplicate bottles in all groups on days 0, 7, 14, 21 and 28
- Sampling method: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. Because of the nitrogen content of the test item, samples for nitrate and nitrite analysis were taken from all vessels (of test item, inoculum control and toxicity control group) and the oxidised nitrogen (nitrate and nitrite) concentrations were measured. The nitrite and nitrate concentrations were determined using photometric method with Nitrite and Nitrate Cell Tests (MERCK®). The nitrate and nitrite analysis of the 7-day, 21-day and 28- day samples was performed directly after oxygen measurements. For technical reason the nitrate and nitrite analysis of the start (day 0) samples was performed together with the 7-day samples on the 7th day of the test and the analysis of 14-day samples was performed seven days later, together with the 21-day samples on the 21st day of the test.
- Sample storage before analysis: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 (+ 2 reserve) bottles containing only inoculum (inoculum control)
- Procedure control: 10 (+ 2 reserve) bottles containing the reference item and inoculum (procedure control)
- Toxicity control: 10 (+ 2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)

Preliminary study:

not performed

Key result

Parameter:

% degradation (O2 consumption)

Value:

36

Sampling time:

28 d

Details on results:

The difference of duplicate values for the biodegradation at the plateau, at the end of the test or at the end of the 10-d window was not greater than 20 %.
The differences between the duplicate biodegradability values at the plateau (and/or at the end of the test) in the test item, procedure control and toxicity control groups were acceptable. From the 14th to 28th day of the test, the highest difference between the duplicate biodegradability values was 10 %, obtained in the test item group, on the 14th day of the test. Higher difference (26 %) was noticed between the duplicate calculated biodegradation values in the test item group on the 7th day of the test. This higher percentage was considered not contradictive with the validity of the study since the test item biodegradability plateau just started on about the 7th day and the difference between the corresponding measured dissolved oxygen concentration values (on the 7th day: 6.04 and 6.52 mg/L) was minimal, about 9 %; therefore, this result does not affect the validity of the study.

Results with reference substance:

The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. (The percentage degradation of the reference item was 72.6 % on the 14th day.)

Table 2: Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Flask	mg O2/L after n days of exposure
	[mg/L]	No.	0	7	14	21	28
Test item	2.0	1a	7.72	6.04	5.17	5.12	4.95
		1b	7.65	5.52	5.30	5.05	4.83
		mean	7.69	5.78	5.24	5.09	4.89
Reference item	3.0	2a	7.90	4.62	3.83	3.51	3.18
		2b	7.76	4.83	3.57	3.33	3.06
		mean	7.83	4.73	3.70	3.42	3.12
Inoculum control	–	3a	7.89	7.83	7.42	7.37	7.28
		3b	7.82	7.52	7.29	7.04	6.84
		mean	7.86	7.68	7.36	7.21	7.06
Toxicity control	Test item: 2.0 Reference item: 3.0	4a	7.70	2.50	2.51	2.04	1.61
		4b	7.64	2.32	2.19	1.89	1.31
		mean	7.67	2.41	2.35	1.97	1.46

 

 

Table 2: Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Flask	mg O2/L after n days of exposure
	[mg/L]	No.	7	14	21	28
Test item	2.0	1a	1.50	2.05	1.95	1.98
		1b	1.95	1.85	1.95	2.03
Reference item	3.0	2a	3.10	3.57	3.74	3.93
		2b	2.75	3.69	3.78	3.91
Toxicity control	Test item: 2.0 Reference item: 3.0	4a	5.02	4.69	5.01	5.30
		4b	5.14	4.95	5.10	5.54

Oxygen depletion: (m_t0 - m_tx) - (m_bo - m_bx), where:

m_t0: oxygen concentration (mg/L) of test group on day 0 (1a, 2a, 4a and 1b, 2b, 4b from Table 1)

m_tx: oxygen concentration (mg/L) of test group on day x (1a, 2a, 4a and 1b, 2b, 4b from Table 1)

m_b0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 1)

m_bx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 1)

 

Table 3: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Flask	Percent of biodegradation after n days of exposure
	[mg/L]	No.	7	14	21	28
Test item	2.0	1a	27.0	36.9	35.1	35.5
		1b	35.1	33.3	35.1	36.4
		mean	31.0	35.1	35.1	36.0
Reference item	3.0	2a	62.0	71.4	74.8	78.5
		2b	55.0	73.8	75.6	78.1
		mean	58.5	72.6	75.2	78.3
Toxicity control	Test item: 2.0 Reference item: 3.0	4a	45.1	42.2	45.1	47.6
		4b	46.2	44.5	45.9	49.8
		mean	45.7	43.4	45.5	48.7

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In a Closed Bottle Test according to OECD TG 301 D after 28 days 36.0 % of the test item were biodegraded (mean, measured based on its theoretical oxygen demand (ThODNH4)). The test item is hence considered to be not readily biodegradable, as the pass level for ready biodegradability of 60 % removal based. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control was higher than 25 % within 14 days.

Executive summary:

A Closed Bottle Test according to OECD TG 301 D was carried out to determine the ready biodegradability of the test item. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 2.0 mg/L.
The test item concentration was chosen based on the on the calculated ThODNH4 value of the test item (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 2.78 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the applied test conditions, no ready biodegradation of this test item was noticed. After 28 days only 36.0 % of the test item was biodegraded (mean, measured based on its theoretical oxygen demand (ThODNH4)). The test item is hence considered to be not readily biodegradable, as the pass level for ready biodegradability of 60 % removal based on the theoretical oxygen demand (ThODNH4) within a 10-day window was not met. Based on the results of the analytical determination of possible nitrite and nitrate development, oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population. Consequently, the biodegradability value of the test item was calculated based on its ThODNH4; any correction, based on the measured nitrite nitrate content was not performed. The reference item, sodium benzoate, was sufficiently degraded to a mean of 72.6 % after 14 days, and to a mean of 78.3 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 43.4 % biodegradation was noted within 14 days and 48.7 % after 28 days of incubation (from about the 7th days of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system). According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control was higher than 25 % within 14 days.

Description of key information

In a Closed Bottle Test according to OECD TG 301 D after 28 days 36.0 % of the test item were biodegraded (mean, measured based on its theoretical oxygen demand (ThODNH4)). The test item is hence considered to be not readily biodegradable, as the pass level for ready biodegradability of 60 % removal based. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control was higher than 25 % within 14 days.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

A Closed Bottle Test according to OECD TG 301 D was carried out to determine the ready biodegradability of the test item. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 2.0 mg/L. The test item concentration was chosen based on the on the calculated ThODNH4 value of the test item (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 2.78 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the applied test conditions, no ready biodegradation of this test item was noticed. After 28 days only 36.0 % of the test item was biodegraded (mean, measured based on its theoretical oxygen demand (ThODNH4)). The test item is hence considered to be not readily biodegradable, as the pass level for ready biodegradability of 60 % removal based on the theoretical oxygen demand (ThODNH4) within a 10-day window was not met. Based on the results of the analytical determination of possible nitrite and nitrate development, oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population. Consequently, the biodegradability value of the test item was calculated based on its ThODNH4; any correction, based on the measured nitrite nitrate content was not performed. The reference item, sodium benzoate, was sufficiently degraded to a mean of 72.6 % after 14 days, and to a mean of 78.3 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 43.4 % biodegradation was noted within 14 days and 48.7 % after 28 days of incubation (from about the 7th days of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system). According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control was higher than 25 % within 14 days.