2-(ethylnitroamino)ethyl nitrate

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:

04 September 2019 - 02 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according to OECD 301F. GLP study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant ”Czajka” , Warsaw, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was aerated for 7 days, at the test temperature of about 22 °C, until application. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids. Before application the sludge was washed in a mineral medium.
- Concentration of sludge: 3.91 g suspended solids/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Remarks:

(44.7 mg/l of organic carbon)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: 10ml of solution (A) in 800 ml water, plus 1 ml solutions (B), (C), (D) and dilution water up to 1 L. The following stock solutions were used, prepared with analytical grade reagents: Solution (A) contains: 8.50 g monopotassium dihydrogen orthophosphate (KH2PO4), 21.75 g dipotassium monohydrogen orthophosphate (K2HPO4), 33.40 g disodium monohydrogen orthophosphate dihydrate (Na2HPO4·2H2O), 0.50 g ammonium chloride (NH4Cl), in 1 L water. Solution (B) contains: 27.50 g calcium chloride, anhidrous (CaCl2) in 1L water. Solution (C) contains: 22.50 g magnesium sulphate heptahydrate (MgSO4·7H2O) in 1L water. Solution (D) contains: 0.25 g iron(III) chloride hexahydrate (FeCl3·6H2O) in 1L water.
- The water used is double-distilled, containing 3.1±0.4 mg/L of organic carbon (< 10% of the organic carbon content introduced by the test item), checked by DOC analysis using spectrophotometer Hach DR 3900 and Hach-Lange reagents.
- Test temperature: 22 ± 1ºC
- pH: 7.4 ± 0.2
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes
- Volume of test solution in flask, V: 0.164 L

TEST SYSTEM
- Number of culture flasks/concentration: triplicates were used, flasks #13, 14, 15 containing test item (100 mg/l) and inoculum (30mg/L SS).
- Measuring equipment: O2 uptake was measured by a closed WTW OxiTop OC 110 repirometer.
- Details of trap for CO2 and volatile organics if used: potassium hydroxide solution.

SAMPLING
- Sampling frequency: O2 uptake data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recorded and stored in the measuring heads of the sample bottles.
- Sampling method: Readings from apparatus (closed WTW OxiTop OC 110 repirometer).

CONTROL AND BLANK SYSTEM
- Inoculum blank: flasks #1, 2, 3, containing only inoculum 30mg/L SS.
- Procedure control: flasks #4, 5, 6, containing reference item (sodium acetate 100 mg/l) and inoculum 30mg/L SS.
- Toxicity control: flasks #16, 17, 18, containing test item, reference item at the same concentrations as in the individual solutions and inoculum 30mg/L SS.
- Other: correction for interference by nitrification:
-Flasks 0b1, 0b2: Test suspension containing test item (100 mg/L) and inoculum at start.
-Flasks 0tox2/1, 0tox2/2: Test suspension containing test item and reference item at the same concentrations as in the individual solutions and inoculum at start.

STATISTICAL METHODS: The calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc., USA purchased from GAMBIT CoiS Ltd, Poland.

Reference substance:

acetic acid, sodium salt

Remarks:

CAS No: 127-09-3, purity p.a. 99.7%, source: CHEMPUR, Piekary Śląskie, Polska.

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

-The biodegradability attained 0.0% within 28 days. The pass level for ready biodegradability is 60% of ThOD.
-The reference item reached 96.9% of biodegradation and the level for ready biodegradability by 4 days
-In the toxicity test the biodegradation was equal to 53.4% in 14 days
-The oxygen uptake of the inoculum blank was equal to 35.8 mg/L in 28 days
-The pH values of flasks were in the range of 7.45-7.66

Results with reference substance:

The reference item reached 96.9% of biodegradation and the level for ready biodegradability by 4 days (>60% reached before day 14).

Table 2. Sample oxygen uptake: biodegradability.

Time, days		1	3	5	7	9	12	14	16	18	21	23	25	28
Test item O2uptake, mg/L	a1	7.3	13.1	17.2	21.0	22.3	26.9	27.9	28.8	29.0	31.0	33.0	34.9	37.5
	a2	7.0	11.1	13.8	15.1	15.9	18.2	17.7	20.1	22.7	25.2	27.9	30.4	33.5
	a3	6.1	13.6	18.0	21.8	23.3	28.4	28.0	30.1	29.7	32.6	34.9	37.5	40.4
	am. avg	6.8	12.6	16.3	19.3	20.5	24.5	24.6	26.3	27.1	29.6	31.9	34.3	37.2
Blank test O2uptake. mg/L	b1	9.3	14.1	16.2	16.9	20.6	24.0	25.2	25.8	28.3	30.5	33.3	34.8	37.9
	b2	8.3	13.3	15.6	18.9	20.2	23.7	25.4	25.0	26.0	27.3	29.5	31.8	33.9
	b3	6.7	12.7	16.1	19.0	19.3	24.8	25.1	27.2	27.0	30.1	31.4	32.7	35.6
	bmavg	8.1	13.4	15.9	18.3	20.1	24.2	25.2	26.0	27.1	29.3	31.4	33.1	35.8
Reference item O2uptake. mg/L	w1	18.3	58.7	70.6	77.2	85.2	93.2	98.3	98.5	101.1	106.9	108.8	110.1	112.9
	w2	21.7	60.4	72.7	80.2	87.9	94.0	99.1	98.6	101.0	103.3	104.5	107.1	109.9
	w3	19.1	60.9	71.8	80.8	87.8	93.8	95.7	98.5	100.0	101.1	104.0	107.1	109.6
	wm. avg	19.7	60.0	71.7	79.4	87.0	93.7	97.7	98.5	100.7	103.8	105.8	108.1	110.8
Toxicity control O2uptake mg/L	tox1	14.4	60.8	71.5	82.3	88.3	96.5	99.9	100.8	102.5	106.2	107.6	108.0	111.4
	tox2	13.7	58.1	71.5	79.5	87.0	92.5	95.7	--	--	--	--	--	--
	tox3	8.7	56.6	71.1	80.9	88.4	93.9	99.1	--	--	--	--	--	--
	tox1-3avg	12.3	58.5	71.4	80.9	87.9	94.2	98.2	--	--	--	--	--	--
Corrected test item O2uptake, mg/L	a1- bm	-0.8	-0.3	1.3	2.7	2.3	2.7	2.7	2.8	1.9	1.7	1.6	1.8	1.7
	a2- bm	-1.1	-2.3	-2.2	-3.1	-4.1	-5.9	-7.5	-5.9	-4.4	-4.1	-3.5	-2.7	-2.3
	a3- bm	-2.0	0.2	2.1	3.5	3.3	4.2	2.8	4.1	2.6	3.3	3.5	4.4	4.6
Reference item %degradation, % BOD/(ThODxC)x100 ThOD = 0.78 mgO2/mg C = 100 mg/L	R1(w1)	13.1	58.2	70.1	75.6	83.5	88.5	93.6	93.0	94.9	99.5	99.2	98.7	98.9
	R2(w2)	17.4	60.2	72.8	79.5	86.9	89.5	94.8	93.0	94.7	94.9	93.7	94.9	95.0
	R3(w3)	14.1	60.9	71.7	80.2	86.9	89.2	90.3	93.0	93.4	92.1	93.1	94.9	94.6
	Rrefavg	15.3	59.2	71.4	77.5	85.2	89.0	94.2	93.0	94.8	97.2	96.5	96.8	96.9

 

Table 3. Correction for oxygen uptake for interference by nitrification. Test item

days	0		28			difference
1) Concentration of nitrate (mg N-NO3/L)	Flask #0b1	Flask #0b2	Flask #13	Flask #14	Flask #15	1.942
	7.09	7.92	9.44	9.49	9.41
	Avg: 7.505 ± 0.587		Avg: 9.447 ± 0.040
2) Oxygen equivalent (4.57× N-NO3) (mg/L)	8.87
3) concentration of nitrite  (mg N-NO2/L)	Flask #0b1	Flask #0b2	Flask #13	Flask #14	Flask #15	difference
	0.012	0.013	0.001	0.002	0.002	-0.01
	Avg: 0.012 ± 0.001		Avg: 0.002 ± 0.001
4) Oxygen equivalent (3.43×N-NO2) (mg/L)	-0.03
5) total oxygen equivalent 2) + 4)	8.84

Table 4. Correction for oxygen uptake for interference by nitrification. Toxicity test

days	0		14		difference
1) Concentration of nitrate (mg N-NO3/L)	Flask #0tox2/1	Flask #0tox2/2	Flask #17	Flask #18	1.41
	7.70	7.78	9.69	8.61
	7.74 ± 0.06		9.15 ± 0.76
2) Oxygen equivalent (4.57× N-NO3) (mg/L)	6.44
3) concentration of nitrite (mg N-NO2/L)	Flask #0tox2/1	Flask #0tox2/2	Flask #17	Flask #18	difference
	0.018	0.012	0.000	0.000	-0.015
	0.015 ± 0.004		0.000 ± 0.000
4) Oxygen equivalent (3.43×N-NO2) (mg/L)	-0.051
5) total oxygen equivalent 2) + 4)	6.39

Table 5. The pH values of test flasks (no adjustment of pH was conducted).

flask #	13	14	15	1	2	3	4	5	6	16	17	18
	Test item			Inoculum blank			Reference item			Toxicity test
initial	7.66	7.64	7.63	7.62	7.62	7.62	7.54	7.60	7.60	7.66	7.60	7.59
final	7.48	7.45	7.47	7.23	7.47	7.40	8.55	8.61	8.63	8.68	8.52	8.52

 

Validity criteria fulfilled:

yes

Remarks:

See "overall remarks".

Interpretation of results:

not readily biodegradable

Conclusions:

Under the ready aerobic biodegradability test, the test item attained 0.0% biodegradation thus it can not be classed as readily biodegradable.

Executive summary:

A ready biodegradability test in an aerobic aqueous medium with manometric respirometry method was performed on the test item, according to OECD 301F / EC C.4 – D manometric respirometry methods, following GLP principles. 100 mg/l of test item was inoculated with activated sludge (30 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at constant temperature (22 ± 0.4ºC) for 28 days. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. Also, as the test item is a nitrogen-containing substance the obtained results for oxygen uptake were corrected for interference by nitrification using additional controls in the test item and toxicity tests. In the toxicity test, containing both the test item and a reference item, on the 14th test day the biodegradation (based on ThOD) attained 53.4%. Thus, the test item is not inhibitory. All validity criteria were fulfilled. The biodegradability of the test item attained 0.0 % within 28 days. The pass level for ready biodegradability is 60% of ThOD. Thus, the test item can not be classed as readily biodegradable.

Description of key information

Key study: Test method according to OECD 301F / EU C.4 (Manometric respirometry methods), GLP study. The test item attained 0.0% biodegradation. Thus, the test item can not be classed as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater