2-(butylnitroamino)ethyl nitrate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 31 August 2017 and 28 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 306 (Biodegradability in Seawater)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(butylnitroamino)ethyl nitrate

EC Number:

279-976-7

EC Name:

2-(butylnitroamino)ethyl nitrate

Cas Number:

82486-82-6

Molecular formula:

C6H13N3O5

IUPAC Name:

butyl(nitro)[2-(nitrooxy)ethyl]amine

Specific details on test material used for the study:

Name : Butyl-NENA
NIVA GLP substance number: G128
CAS : 82486-82-6
Batch number : 02/16, alternative 160002
Appearance : Yellow liquid, oily
Purity : 100 %. Added stabilizer n-Methyl p-Nitro Aniline, 0.5 %
Solubility in water : 100-500 ppm.
ThODNH4 : 0.70 mgO2/mg ThODNO3: 1.62 mgO2/mg

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: marine

Details on inoculum:

The seawater was pumped from a depth of 60 meter in the Oslofjord, from NIVA's Research Station at Solbergstrand, and collected in 30 L polyethylene containers. The temperature is logged at the research station and not measured according to GLP.

Appearance: Clear
Salinity: 35‰
Temperature: 8.1 ˚C

Dissolved organic carbon concentration was analyzed after storage (1.1 mg C/L). The concentration of heterotrophic bacteria was also determined (1.3x105 CFU/mL) by plating on marine agar and incubated for 8 days at 25 ± 2 °C.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

ca. 3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation 3

Parameter followed for biodegradation estimation:

other: Nitrification

Details on study design:

Preparation of seawater test medium
The seawater was transported to NIVA in Oslo and stored at test temperature for three days in the dark. Water (15 L) was siphoned into new can and aerated for 1.5 hours. Nutrient stock solutions A, B, C and D (OECD 306) were added (1 ml/L). The composition of the stock solutions is shown in Table 1.

Preparation of test bottles
Blank control
Test bottles (15 pc) were filled by siphoning from the seawater test medium container and sealed with a glass stopper.

Test sample bottles
Test substance was added to the test bottle by using an inert support technique (ISO 10634): approximately 0.9 mg butyl-NENA was added to glass cover slip, weight measured exactly on balance, and added to the empty test bottles. The bottles (15pc) where filled carefully with seawater, allowing minimum overflow when adding the glass stopper. The weight of the test bottles was measured before and after addition of test water to calculate the exact volume in each bottle.

Reference compound bottles (test control)
A stock solution of aniline (C6H5NH2, 99 %, Mw 93.13) was prepared as the reference (test control) by dissolving 0.2722 g aniline in 100 ml MilliQ water. A container was filled with 5 L seawater test media by siphoning and added 5 ml aniline stock solution and mixed. Test bottles (12 pc) were filled by siphoning from this container and sealed with a glass stopper.

Toxicity control
Bottles for toxicity control where added test substance as described for test bottles, and filled with the same stock of seawater media added aniline as described for the reference bottles.

Incubation and sampling
All bottles (blank, test, reference and toxicity) were incubated under dark plastic in a temperature controlled room (21.2 ± 1.6oC).

The dissolved oxygen (DO) concentration was measured on days 0, 7, 14, 21 and 28.

Both Butyl-NENA and aniline are nitrogen containing compounds and the ThOD depends on whether the nitrogen is degraded to ammonium or oxidized further to nitrite and nitrate. The concentrations of nitrite and nitrate were measured in all bottles after DO-measurements.

Physical and chemical measurements
Chemical analysis

• DOC: Tekmar Dohrmann Apollo 9000 HS using NIVA method G5-3 (based on modified NS-EN 1484:1997)
• Nitrate and nitrite: Skalar San Plus Autoanalysator using NIVA method D 3-3 (based on modified NS-4745).

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

Key result

Parameter:

% degradation (DOC removal)

Value:

11

Sampling time:

28 d

Details on results:

Temperature
Temperature range during test was maximum 22.7”C and minimum 19.6”C, resulting in a mean test temperature of 21.2 ± 1.6”C

BOD5 / COD results

BOD5 / COD

Key result

Parameter:

ThOD

Value:

11 other: %

Results with reference substance:

Aniline was used as the reference compound in the test to assess the viability of the indigenous bacteria used to evaluate the biodegradability of the test compound. The biodegradability of this reference compound should be at least 60% of the reference material Aniline (i.e. >60% of its theoretical oxygen demand; ThOD) within 28 days and at least 50% within a timeframe of 2-12 days ending latest at day 19 after commencing the test as according to a EEC ring-test (Nyholm and Kristensen).

The biodegradation of aniline in this experiment was 63% after 7 day’s incubation and 70% after 14 days (ThODNH4). No nitrification occurred. The criteria of 50% degradation within 12 days and >60% within 28 days are fulfilled.

Any other information on results incl. tables

Nitrification assessment

Because both Butyl-NENA and Aniline are nitrogen containing substances, the possibility of nitrification must be assessed by comparing the concentration of nitrite + nitrate during the incubation period.

 

No increase in nitrite + nitrate was observed in the blank or the reference controls. The test bottles with Butyl-NENA and the toxicity control had a higher starting concentration of nitrate and nitrite than the blank, however, there is no significant increase of the average nitrite + nitrate concentration in the test bottles compared to the starting concentration even if some of the bottles had higher concentrations.

Validity of test

Blank control

The dissolved oxygen in the blank control bottles decreased 6% (from 7.20 mg/L to 6.74 mg/L) over the 28 days’ test period. This is within the acceptable limit of 30%.

 

Toxicity control

The oxygen consumption in the toxicity control was comparable to the sum of the oxygen consumption from the reference control and the test substance separately, confirming no inhibition of the bacteria from Butyl-NENA.

Biodegradation of the test samples

The test substance Butyl-NENA had very little biodegradation in this test, with 11% reduction of ThOD after 28 days incubation assuming no nitrification.

Table1: Nitrification in test, reference control and toxicity control

	NO2+NO3 concentration [µg N/L]
Sample	Initial conc.	d7	Average ± SD	d14	Average ± SD	d21	Average ± SD	d28	Average ± SD
Blank	131	126	131 ± 5	116	118 ± 4	121	121 ± 2	109	111
		135		122		120		6
		132		115		123		113
G128	149	195	197 ± 8	175	180 ± 13	170	173 ± 3	175	157 ± 27
		205		195		175		170
		190		170		175		126
Ref	127	130	130	118	124	108	116
		130		130		123
Tox	180	195	200	175	175	170	173
		205		175		175

Table 2 ThOD and Oxygen Consumption

	ThODNH4
G128	0,70
day	0	7	14	21	28
Oxygen consumption	0	0,17	0,22	0,22	0,22
% of ThOD NH4	0	8 %	10 %	10 %	11 %
	ThODNH4
Reference	2,41			concentration	2,722	mg/L
day	0	7	14	21
Oxygen consumption	0	4,13	4,62	4,62
% of ThOD NH4	0	63 %	70 %	71 %
Tox
day	0	7	14	21
Oxygen consumption	0	4,23	4,64	4,95
% of ThOD NH4	0	49 %	52 %	57 %

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The results of the study indicate that Butyl-NENA (3 mg/L) was 11% biodegraded in seawater over an incubation period of 28 days.

Executive summary:

The biodegradability of Butyl-NENA has been assessed in seawater at 21.2 ± 1.6˚C using the Closed Bottle Test method according to OECD Guidelines for Testing of Chemicals 306 (OECD, 1992) modified for addition of poorly soluble substances. 

 The test compound was exposed to indigenous microorganisms in natural seawater.Butyl-NENAwas tested at a concentration of 3.1 ± 0.1 mg/L, corresponding to a ThOD of 5.0 mgO₂/L.

 The biodegradation of Butyl-NENA in this test was 11% after 28 days, assuming full nitrification. No inhibitory effect of the microorganisms was assessed during the study.

 The reference control, aniline, reached 63% degradation within seven days and thus confirmed the viability of the microorganisms in the seawater used.