2,2'-[oxybis(2,1-ethanediyloxy)]bisacetic acid

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:

from 2017-10-03 to 2017-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/batch No(s): 03/2016_R_JM

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge, microorganisms from a domestic waste water treatment plant. Supplied on 03 October 2017 (seven days before the main test).
- Storage conditions: aerating (2 L/minute) of the activated sludge in mineral medium at 20.0 - 21.8 °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 and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with test water (mineral medium) and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.39, just before use: 7.24.
- Pretreatment: The inoculum was not pre-adapted to the test chemical.
- Concentration of sludge: 5 g dry material per litre
- Initial cell/biomass concentration: ~1E8/L
- Filtered: yes
- Type and size of filter used: cotton wool

Duration of test (contact time):

28 d

Initial conc.:

5.75 mg/L

Based on:

ThOD

Initial conc.:

5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium used. Prepared according to OECD 301.
- Test temperature: 20.0 - 20.1 °C
- pH: 7.38
- pH adjusted: no
- Aeration of dilution water: yes, for 20 minutes
- Suspended solids concentration: 5 g dry material/L
- Continuous darkness: yes
- Oxygen concentration of the test water: About 8 - 9 mg/L (was measured at the start of the test and found to be 8.67 mg/L).

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
- Number of culture flasks/concentration:
10 (+ 2 reserve) bottles containing the test item and inoculum;
16 (+ 2 reserve) bottles containing the reference item and inoculum (procedure control);
- Measuring equipment: The oxygen concentration was measured with an O2 electrode.

SAMPLING
- Sampling frequency: in all duplicate bottles in all groups on days 0, 7, 14, 21 and 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 16 (+ 2 reserve) bottles containing only inoculum (inoculum control)
- Toxicity control: 10 (+ 2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)

Reference substance:

benzoic acid, sodium salt

Remarks:

ThOD 5.01 mg O2/L

Preliminary study:

No toxic effect of the test item was found at the investigated concentration - 5.0 mg/L.

Key result

Parameter:

% degradation (O2 consumption)

Value:

21.2

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of the test item reached a mean of 21.2 % after 28 days based on its ThOD.
The pass level for ready biodegradability is the removal of 60 % ThOD in a 10-day window. Minimal biodegradation of the test item occurred in this study; the value remained far below the pass level; therefore, the test item was considered to be not ready biodegradable.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 84.0 % after 14 days, and to a mean of 90.7 % after 28 days of incubation, based on its ThOD. The biodegradability reached its plateau on about the 14th day.

Biodegradation of Toxicity Control

In the toxicity control containing both, the test item and the reference item, a mean of 40.5 % biodegradation was noted within 14 days and a mean of 41.6 % biodegradation was determined after 28 days of incubation.

Validity of the Study

Inoculum Control: The oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days. It was 1.36 mg O2/L in average.

Oxygen Concentration: The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. (The lowest value was 3.12 mg O2/L, it was measured on the 28th day in the procedure control.)

Parallels: The difference of duplicate values for the degradation at the plateau, at the end of the test or at the end of the 10-d window was not greater than 20 %. The highest difference (12.6 %) between the duplicate values from the 14th to the 28th day of the test (this period was taken into consideration as approximately biodegradation plateau) or at the end of the test for degradation was calculated in the test item group, observed on the 21st day of the test.

Reference Item: 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 84.0 % on the 14th day.)

Results

Table 1.:    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	5.0	1a	9.33	7.66	7.13	6.77	6.69
		1b	9.33	7.71	7.02	6.92	6.81
		mean	9.33	7.69	7.08	6.85	6.75
Reference item	3.0	2a	9.27	4.69	3.65	3.57	3.12
		2b	9.29	4.23	3.78	3.71	3.65
		mean	9.28	4.46	3.72	3.64	3.39
Inoculum control	–	3a	9.21	7.96	7.96	7.91	7.81
		3b	9.24	7.88	7.76	7.96	7.92
		mean	9.23	7.92	7.86	7.94	7.87
Toxicity control	Test item: 5.0 Reference item: 3.0	4a	9.33	3.96	3.45	3.42	3.25
		4b	9.32	4.00	3.33	3.30	3.29
		mean	9.33	3.98	3.39	3.36	3.27

 

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	5.0	1a	0.36	0.83	1.27	1.28
		1b	0.31	0.94	1.12	1.16
Reference item	3.0	2a	3.28	4.26	4.41	4.79
		2b	3.76	4.15	4.29	4.28
Toxicity control	Test item: 5.0 Reference item: 3.0	4a	4.07	4.52	4.62	4.72
		4b	4.02	4.63	4.73	4.67

 

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.:    BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Flask	BOD after n days of exposure
	[mg/L]	No.	7	14	21	28
Test item	5.0	1a	0.07	0.17	0.25	0.26
		1b	0.06	0.19	0.22	0.23
Reference item	3.0	2a	1.09	1.42	1.47	1.60
		2b	1.25	1.38	1.43	1.43
Toxicity control	Test item: 5.0 Reference item: 3.0	4a	0.51	0.56	0.58	0.59
		4b	0.50	0.58	0.59	0.58

 

BOD = (mg O2 of T.i. and/or R.i. – mg O2 of i.control) / mg T.i. and/or R.i. in flask = mg O₂/mg T.i and/or R.i.

where:

T.i.           =  test item

R.i.           =  reference item

i.control    =  inoculum control

 

Table 4.:    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	5.0	1a	6.3	14.5	22.0	22.2
		1b	5.5	16.4	19.4	20.1
		mean	5.9	15.4	20.7	21.2
Reference item	3.0	2a	65.5	85.1	88.2	95.8
		2b	75.1	82.9	85.8	85.6
		mean	70.3	84.0	87.0	90.7
Toxicity control	Test item: 5.0 Reference item: 3.0	4a	36.0	40.0	41.0	41.8
		4b	35.6	41.0	41.9	41.4
		mean	35.8	40.5	41.4	41.6

Biodegradation % = (BOD (mg O2/mg T.i. or R.i.) / ThOD (mg O2/mg T.i. or R.i.)) * 100

where:

T.i.           =  test item

R.i.           =  reference item

ThOD of test item = 1.15 mg O₂/mg test item

ThOD of reference item = 1.67 mg O₂/mg reference item

The biodegradation in the toxicity control was calculated according to the following formula:

 (BOD (mg O2/mg T.i. or R.i.) / [ThOD (mg O2/mg T.i. ) + ThOD (mg O2/ mg R.i.)] * 1/2) * 100

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, fulfilling specific criteria

Conclusions:

The test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window. The total degradation after 28 days of exposure was 21.2 %. The test item is regarded as inherently biodegradable.

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 Closed bottle test according to OECD 301D, Regulation (EC) No. 440/2008 Method C.4 -E and EPA OPPTS 835.3110 and under GLP. The test item was tested at a concentration of 5 mg/L in triplicates, corresponding to an oxygen content of 5.75 mg O2/L. The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The biodegradation of the test item came to 21.2 % after 28 days. In conclusion, the test item is not readily biodegradable as the pass level of 60 % degradation was not reached in a 10-d-window within the 28 days period of the study.

Description of key information

The test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window. The total degradation after 28 days of exposure was 21.2 %. The item is regarded as inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Additional information

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Closed bottle test according to OECD 301D, Regulation (EC) No. 440/2008 Method C.4 -E and EPA OPPTS 835.3110 and under GLP. The test item was tested at a concentration of 5 mg/L in triplicates, corresponding to an oxygen content of 5.75 mg O2/L. The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The biodegradation of the test item came to 21.2 % after 28 days. In conclusion, the test item is not readily biodegradable as the pass level of 60 % degradation was not reached in a 10-d-window within the 28 days period of the study.