2-(butylnitroamino)ethyl nitrate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conduced between 25 January 2018 and 01 February 2018

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:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

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:

Identification Butyl-NENA
NIVA substance number G128
CAS No. 82486-82-6
Batch No. 160002
REACH registration No. Not stated
EC No. Not stated
Purity 100 %. Added stabilizer n-Methyl p-Nitro Aniline, 0.5 %
Substance appearance Yellow liquid, oily
Date received 18.05.2017
Storage conditions Closed in bottle, dry at ambient temperature
Expiry data June 2021
Stability Stable under normal temperature conditions and recommended use. Explosive when mixed with oxidizing substances.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Remarks:

Elendt M7 medium

Details on test solutions:

Preparation of test solutions
A stock solution of 100 mg/L, the highest test concentration in the study, were prepared by adding 500 ml M7 to 50.06 mg butyl-NENA. This solution was also used to prepare the remaining test concentrations as indicated in the following table.

Concentration (mg/L) Volume stock solution (ml) Volume medium (ml) Final volume (ml)
Control 0 250 250
1 2.5 247.5 250
3.2 8 242 250
10 25 225 250
32 80 170 250

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Name Daphnia magna
Source In house cultures at NIVA (DHI – clone)
Culture details The Daphnia are cultured in Elendt M7 media under the same conditions as used in the test according to a standard operating procedure. Cultures are fed with Psedukirchineriella subcapitata.
Life-stage/Age Animals used for the test were young daphnia, less than 24 hours old
Justification Daphnia magna were used as the test organism as they are easily cultured within the laboratory and are recommended for use in freshwater ecotoxicity assessments [1].

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

20 ± 2”C

pH:

7 ± 1

Details on test conditions:

Exposure conditions
The exposure vessels were placed in a climate controlled room set at 20ºC with a photoperiod providing 16 hours light: 8 hours dark. Daphnids were not fed during the exposure period.

Addition of test animals
Offspring of D. magna (DHI-clone) were separated from in house cultures and were less than 24h old at the initiation of the test. The test animals were randomly selected and added to each test vessel using a wide bore pipette. Care was taken to minimise the amount of medium transferred (< 2 ml per replicate added). Twenty animals, 5 per replicate (4 replicates per concentration) were added per concentration.

Test procedure and Observations
All test vessels were rinsed with appropriate test solution prior to being filled for the test. After preparation of the test solutions and rinsing of test vessels, the test solutions were dispensed into the test vessels to give approximately 40 ml of solution per replicate. Test solutions were dispensed moving up the concentration gradient and starting with the control. The test vessels were identified by study number, concentration, and replicate number.

The study was initiated with the addition of the daphnids. At 24 hour intervals, observations were made for immobilisation and these data were recorded in the study book. Immobilisation is defined as the inability of the organism to swim within 15 seconds after gentle agitation of the test vessel, even if movement of antennae is observed. The daphnids were not fed during the study. At the start and end of the test, samples were removed for physicochemical analysis.

Physicochemical analysis
The pH was measured with an ORION STAR A211 pH meter in the control and highest test solution at the start of the test and after 48h.

Temperature was measured using min./max. thermometer with the sensor placed in water at the level of the test vessels. Temperature was recorded at the beginning and the end of the test.

Dissolved oxygen was measured with an InoLab OXI 7310 meter in the control and highest test solution at the start of the test and after 48h.

At the start of the test measurements were made on excess test solution, measurements at the end of the exposure were on solution from one replicate.

Data evaluation
For each test concentration and the control, the data from the replicates were pooled and percentage of immobilisation after 24 and 48h calculated (Table 2). The raw data was imported to ToxRatPro© (Version 3.2.1) where the OECD 202 template was used for the statistical analysis.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Key result

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

96 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 6

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Validity criteria
The test met all the validity criteria (Table 1) required by OECD guidelines [1], and is considered valid. (Table 1).

Physicochemical parameters
The physicochemical parameters are showed in Table 2.

Data evaluation
No immobility was observed in the controls at 24h or 48h exposure. After 24h of exposure, immobility was observed for 1 of the 20 D. magna exposed to 32 mg/L, whereas 11 of the 21 D. magna exposed to 100 mg/L were immobile after 24h (Table 3). After 48h of exposure, 14 of 20 D. magna exposed to 32 mg/L were immobile, whereas 15 of 21 of the daphnia exposed to 100 mg/L were immobile after 48h. The results of the study are summarised in Table 4.

Any other information on results incl. tables

Table1. Summary of results for validity criteria

Parameter	Criterion	Observed
Dissolved oxygen (at end of test)	≥ 3 mg/L	8.82-10.26 mg/L
Percentage mortality in the control	≤ 10%	0 %

Table2. Physico-chemical parameters

Concentration	pH		Dissolved oxygen (mg/L)		Temperature (°C)
mg/L	start	end	start	end	start	end
Control	8.04	7.92	8.96	10.26	19.9-21.1	19.3-21.3
100	7.85	7.96	8.82	10.25

Table3. Survival data for D. magna at 24 and 48 hours after exposure toButyl-NENA

Test substance:                                          Butyl-NENA
Concentration (mg/L)		24 hour (survival)					48 hour (survival)					Survived/added daphnia			% mortality
		1		2		3		4		1		2		3		4		24 h		48 h		24 h		48 h
Control *		5		5		5		5		5		5		5		5		20/20		20/20		0		0
1		5		5		5		5		5		5		5		5		20/20		20/20		0		0
3.2		5		5		5		5		5		5		5		4 (1)		20/20		19/20		0		5
10		5		5		5		5		5		5		5		5		20/20		20/20		0		0
32		4 (1)		5		5		5		0 (5)		0 (5)		4 (1)		2 (3)		19/20		6/20		5		70.0
100		2 (3)		3 (2)		2 (3)		3 (3)		1 (4)		2 (3)		1 (4)		2 (4)		10/21		6/21		52.4		71.4

*Shared control with study 17159/6 and 17159/7

Table 4. Summary of results

Test substance		Butyl-NENA
Endpoint		Concentration (mg/L)		95 % Confidence intervals
24h NOEC		32		NA
24h LOEC		100		NA
24h EC50		96		70-174
48h NOEC		10		NA
48h LOEC		32		NA
48h EC50		36		nd

Where: NOEC = No observed effect concentration based on nominal loading.

LOEC = Lowest observed effect concentration based on nominal loading.

EC₅₀represents the loading rate that results in 50 % immobilisation.

NA = Not applicable.

nd – not determined due to mathematical reasons

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of the test substance for 24h and 48h exposure was 96 mg/L (24h EC50) and 36 mg/L (48h EC50). The NOEC and LOEC for immobility after 24h of exposure to butyl-NENA were 32 mg/L (NOEC) and 100 mg/L (LOEC). The NOEC and LOEC for immobility after 48h of exposure to butyl-NENA were 10 mg/L (NOEC) and 32 mg/L (LOEC).

Executive summary:

The acute toxicity of Butyl-NENA to the freshwater crustaceanDaphnia magnawas investigated. The test was performed according to OECD 202 (2004)Daphnia sp.Acute immobilisation test [1]. The test animals were young daphnia, less than 24h old, from in house cultures (DHI clone) at NIVA.

A range of concentrations (1, 3.2, 10, 32, and 100 mg/L) were prepared in Elendt M7 medium. The M7 medium control was shared with study 17159/6 and 17159/7. Four replicate vessels, each containing fiveD. magnawere employed for the control and each treatment. Immobility was assessed at 24h and 48h.

The objective of the study was to determine the median effective concentration (EC50) of the test substance for 24h and 48h exposure, which was 96 mg/L (24h EC50) and 36 mg/L (48h EC50). The results are summarised as follows:

 

Test substance	Butyl-NENA
Endpoint	Concentration (mg/L)	95 % Confidence intervals
24h NOEC	32	NA
24h LOEC	100	NA
24h EC50	96	70-174
48h NOEC	10	NA
48h LOEC	32	NA
48h EC50	36	nd

Where: NOEC = No observed effect concentration based on nominal loading.

LOEC = Lowest observed effect concentration based on nominal loading.

EC₅₀represents the loading rate that results in 50 % immobility.

NA = Not applicable.

nd – not determined due to mathematical reasons