Reaction products of 2-[[2-(dimethylamino)ethyl]methylamino]ethanol di(hexanoate) and ethyl oxirane

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:

July to October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP principles

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Inoculum of the aqueous phase of non-adapted activated sludge is derived from the municipal sewage treatment plant at Hildesheim, Germany. Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste. The activated sludge was washed twice with chlorine free 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 hours and 10 minutes and 2 hours and 35 minutes respectively. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air until test start. 10 mL/L and 20 ml/L were used to initiate inoculation respectively.

Colony forming units Approx. 10E-7 - 10E-8 CFU/L
in the test vessels

Duration of test (contact time):

28 d

Initial conc.:

25 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Based on the calculated oxygen demand, the test concentration of 25 mg/L, corresponding to an oxygen demand of 52.3 mg O2/L (58.3 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) or were prepared directly in brown glass bottles (test item, toxicity control):
• two for the inoculum control (C1, C2)
• one for the functional control (R)
• two for the test item (P1, P2)
• one for the toxicity control (T1)

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 was filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask.
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 10 mL/L 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.
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 10 mL/L inoculum, were added. 250 mL of this solution was filled in the brown glass bottle of the test item replicates, using a 250 mL measuring flask.
For the toxicity control, the test item and reference item were weighed out and transferred into a measuring flask, the required volumes of mineral medium stock solutions, ultrapure water and 10 mL/L inoculum, were added. 250 mL of this solution were filled in the brown glass bottle of the toxicity control, using a 250 mL measuring flask.
A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles were closed with OxiTop measuring heads and the measuring system was activated.
Temeprature was maintained at 21.4 – 21.8°C (21.4 – 21.6°C second test). The solutions were stirred continously and were kept dark in an incubator.
At test start the pH-values of the prepared solutions in the measuring flasks (inoculum control, functional control and test item) and the prepared solutions in the test vessels (tox-icity control), respectively, were determined. At test end the pH-values of the test solutions in the brown glass bottles were determined.

Reference substance:

benzoic acid, sodium salt

Test performance:

The mean oxygen depletion in the inoculum control was 30.3 mg O2/L and 24.0 mg O2/L (second study) on day 28.

• The percentage degradation of the functional control reached the pass level of 60 % in both studies within 3 days (validity criterion: 60 % after 14 days).
• The mean oxygen depletion in the inoculum control was 30.3 mg oxygen/L on day 28 in the first and 24.0 mg oxygen/L in the second study (validity criterion: < 60 mg oxygen/L).
• The pH-value of all test replicates was in the range of 6.0 - 8.5 at the end of the test.
• The percentage degradation of the toxicity control reached the pass level of 25 % in both studies within 2 days (validity criterion: 25 % after 14 days).
• The difference of extremes of replicate values of removal of the test item at the end of the test was more than 20 % (27 % and 30 %, respectively, on day 28). It is assumed, that some inherent properties of the test item, minimal differences in the bacterial composition of the inoculum inoculum and differences in the pro-gress of adaptation (during the studies) for the different test item constituents are decisive for the outcome of the biodegradation of the test item, resulting in a dif-ference > 20% between the test item replicates. Therefore, the two studies can be regarded as reliable. The higher difference of extremes was considered to have no impact on quality and integrity of the study.

Parameter:

% degradation (O2 consumption)

Value:

45

Sampling time:

28 d

Remarks on result:

other: first study

Parameter:

% degradation (O2 consumption)

Value:

58

Sampling time:

28 d

Remarks on result:

other: second study

Results with reference substance:

Results of the Functional Control for both tests:

The pass level for ready biodegradation (> 60 % degradation) was reached within 3 days. The validity criterion that the degradation should be > 60 % after 14 d was fulfilled.

Results of the Toxicity Control (1st study:

In the toxicity control the biodegradation achieved 68 % after 14 days. After 28 days the biodegradation came to 75 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled

Results of the Toxicity Control (2nd study):

In the toxicity control the biodegradation achieved 66 % after 14 days. After 28 days the biodegradation came to 81 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled

Biodegradation [%] of the Test Item in comparison to the Functional Control and Toxicity Control in the First Study

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item 25mg/L	1	37	34	31	31
	2	43	49	54	58
Functional Control 45 mg/L		79	83	81	80
Toxicity Control 25mg/L Test Item + 45 mg/L Reference Item		61	68	71	75

Biodegradation [%] of the Test Item in comparison to the Functional Control and Toxicity Control in the second study

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item 25mg/L	1	39	55	66	73
	2	34	42	45	43
Functional Control 45 mg/L		72	82	93	97
Toxicity Control 25mg/L Test Item + 45 mg/L Reference Item		55	66	75	81

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item is classified as not readily biodegradable within the test period of 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 according to OECD 301F. The test item concentration selected as appropriate was 25 mg/L, corresponding to a ThOD of 52.3 mg O2/L per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.

 

Summary of the First Study

The mean oxygen depletion in the inoculum control was 30.3 mg O2/L on day 28.

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 84 % degradation on day 15.

In the toxicity control containing both test and reference item 68 % degradation occurred within 14 days. After 28 days the biodegradation came to 75 %. The degradation of the reference item was not inhibited by the test item.

For the test item both replicates reached the 10 % level (beginning of biodegradation) within 2 days. The 60 % pass level was not reached until test end. The mean biodegradation on day 28 was 45 % (1st replicate 31 % and 2nd replicate 58 %). Due to a diffenrence of more than 20% beween the test item replicates, a second test was conducted.

 

Summary of the second study

 

The mean oxygen depletion in the inoculum control was 24.0 mg O2/L on day 28.

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 97 % degradation on day 27.

In the toxicity control containing both test and reference item 66 % degradation occurred within 14 days. After 28 days the biodegradation came to 81 %. The degradation of the reference item was not inhibited by the test item.

For the test item both replicates reached the 10 % level (beginning of biodegradation) within 1 day. The 60 % pass level was reached by the first replicate on day 18 and was missed by the second test item replicate. The mean biodegradation on day 28 was 58 % (first replicate 73 % and second replicate 43 %).

 

The repeatedly divergent results for the test item replicates indicate that – although soluble – the test item seems to have inherent properties that only under ideal conditions allows for biodegradation within the “ready” limits. Therefore, it is assumed, that these inherent properties of the test item, minimal differences in the bacterial composition of the inoculum and differences in the progress of adaptation during the studies for the different constituents are decisive for the outcome of the biodegradation of the test item, resulting in difference > 20 % between the test item replicates. Nevertheless, the test item has to be attributed a greater potential of biodegradability than the classification as “not readily biodegradable” reflects.

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 according to OECD guideline 301 F.
Due to a difference of more than 20% between the test item replicates at the end of test a second test was conducted. In both tests the functional control reached the pass level of >60% within 3 days and the degradation of the reference item was not inhibited by the test item.
In teh first test both test item replicates reached the 10 % level (beginning of biodegradation) within 2 days. The 60 % pass level was not reached until test end. The mean biodegradation on day 28 was 45 % (1st replicate 31 % and 2nd replicate 58 %).
In teh second test both test item replicates reached the 10 % level (beginning of biodegradation) within 1 day. The 60 % pass level was reached by the first replicate on day 18 and was missed by the second test item replicate. The mean biodegradation on day 28 was 58 % (first replicate 73 % and second replicate 43 %).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The repeatedly divergent results for the test item replicates indicate that – although soluble – the test item seems to have inherent properties that only under ideal conditions allows for biodegradation within the “ready” limits. Therefore, it is assumed, that these inherent properties of the test item, minimal differences in the bacterial composition of the inoculum and differences in the progress of adaptation during the studies for the different constituents are decisive for the outcome of the biodegradation of the test item, resulting in difference > 20 % between the test item replicates. Nevertheless, the test item has to be attributed a greater potential of biodegradability than the classification as “not readily biodegradable” reflects.