2-(butylnitroamino)ethyl nitrate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 11 December 2017 and 15 December 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

2012

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 Product Information:
Product Name Butyl-NENA
Batch number 02/16 (alternative160002)
CAS number 82486-82-6
NIVA substance number G128
Assay 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 Butyl-NENA can be stored for at least five years when stored according to recommended storage conditions; dry, below room temperature in unopened original packaging.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The test solutions of Butyl-NENA were prepared at five concentrations (1, 3.2, 10, 32 and 100 mg/L), plus a control in TEst Medium.
The test medium used was ISO 8692 (2) and the composition is in Table 1

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Name Pseudokirchneriella subcapitata
Strain NIVA strain CHL 1
Source NIVA culture collection
Stock culture Cultured in filtered medium ISO 8692 (Appendix 1) at continuous light and at approximately 20oC.
Inoculation culture Inoculum culture was set up 3 days before test initiation in the same medium as used in the test. Incubation conditions were the same as during the test (See 3.6).
Justification Pseudokirchneriella subcapitata were used as the test organism as they are easily cultured within the laboratory and are recommended for use in freshwater ecotoxicity tests (1).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22 ± 2”C

pH:

7.87 - 8.18

Nominal and measured concentrations:

Nominal

Details on test conditions:

Experimental design
The test solutions of Butyl-NENA were prepared at five concentrations (1, 3.2, 10, 32 and 100 mg/L), plus a control. Algal inoculum was added to the test solutions so that the final volume was 50 ml for each concentration of test substance and 100 ml for control, each one with a cell density of approximately 5 x10^6 cells/L. Blanks (with no addition of alga) were also made to verify if the exposure solutions without algae had any interference on the fluorescence readings. The toxicity test was performed with three replicates of each test concentration and six control replicates with test medium. The exposure was performed in 25 ml glass vials, covered with lids to avoid evaporation and the entry of dust into the test solutions. The test volume was 15 ml. The test system was identified by study number, concentration and replicate.

Exposure conditions
The flasks were placed in an orbital shaker incubator (Innova 2, 44R) with continuous agitation. The incubation temperature was set to 22 ± 2”C with continuous illumination from day light-type fluorescent tubes, above the culture vessels and providing direct irradiation.

Test procedure and Observations
The pH was measured with an ORION STAR A211 pH meter in all test solutions and control at the start of the test and in 1 replicate after 72 hours.

Cell counting was performed after 24 ± 2, 48 ± 2 and 72 ± 2 hours, using Cytofluor 2300 to measure fluorescence, at an excitation of 485 nm and emission of 685 nm. 96-well microplates (NUNC®, Oslo, Norway) were used for measuring fluorescence, where 200 µl of each algal sample was pipetted into each well. Wells with just media and test solutions without algae were added to act as blanks. Extra wells with different algal concentrations (in the last row of the same plate and in an extra plate) were read at each time point for determination of the conversion factor between fluorescence readings and cell counts.

Temperature during incubation was recorded on a min./max. thermometer with the sensor placed in water at the level of the test cultures.

The light level was measured at the start and end of incubation using a Dual Display Traceable light meter.

Data evaluation
The conversion factor between the fluorescence measurements (from the Cytoflyor 2300) and cell density (measured by the Coulter Multisizer) was calculated to calibrate the obtained fluorescence data. A linear regression between the fluorescence readings and respective cell counts data was used, to transform the obtained fluorescence data into cell counts.

Growth rate calculations
The growth rate (µ, d-1) for each concentration was calculated from initial cell concentration and cell concentration at each time point (24, 48 and 72 hours) using the statistical program ToxRatPro© (Version 3.2.1)

Effect concentrations (EC)
The effect concentrations on growth: EC50 - effective concentration for 50% reduction, were calculated using ToxRatPro© (Version 3.2.1) for each time point (24, 48 and 72 hours).

No observed effect concentration (NOEC) and lowest observed effect concentration (LOEC)

ToxRatPro© (Version 3.2.1) was also used to determine the NOEC and LOEC after 24, 48 and 72 hours exposure

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Validity criteria
The test met all the validity criteria (Table 1), so it is considered valid.

Physicochemical parameters
The following pH ranges were recorded during the test:
Min: 7.87, Max: 8.18.

The following temperatures were recorded during the test:
Min: 22.0 ºC, Max: 22.3 ºC.

The following light ranges were recorded at the start of the study:
Min: 2180 lux, Max: 2280 lux.
Using the conversion factor of 0.029 from lux to µmol.s-1.m-2 (3) for the GRO-lux lights (as provided in the Innova 44R photosynthetic light bank), Min - Max range at the start of the test in the incubator was:
Min: 63.22 µmol.s-1.m-2, 66.12 µmol.s-1.m-2.
The light was also measured at the end of the study, with the following measurements: Min: 2090 lux (60.61 µmol.s-1.m-2), Max: 2140 lux (62.06 µmol.s-1.m-2).

Algal growth
The fluorescence readings of the blanks show that there was no interference of the exposure solutions in the fluorescence measurements, as there was no significant variation in the fluorescence measurements along time exposure. Fluorescence readings of different cell counts were also measured along time exposure. This data was used to calculate a linear regression between fluorescence and cell counts. The linear regression equation (y=1.7x-183.71; R2=1) was used to transform the fluorescence data into cells counts. Cell counts data was then applied in the statistical program ToxRatPro© for evaluation.

The summary of results from the statistical analyses EC50, NOEC and LOEC for Butyl-NENA are in Table 2.
The growth rate in Pseudokirchneriella subcapitata as dependent test substance Butyl-NENA concentration and time is shown in Table 3

Any other information on results incl. tables

Table 1. Summary of results from the validity criteria.

Parameter	Criterion	Observed
Cell increase in controls after 72 h compared to start	>16 times	140.1 times
Variation coefficient in control	<7%	2.7%
Variation coefficient in section-by-section	<35%	25.9%

Table 2.Summary of EC₅₀, NOECs and LOECs at each time point regarding the growth rate for the tested compound.

	Test substance Butyl-NENA
	24 h	48 h	72 h
EC50	n.d.	66 mg/L	52 mg/L
EC5095%-CL lower	n.d.	39 mg/L	34 mg/L
EC5095%-CL upper	n.d.	108 mg/L	78 mg/L
LOEC	≤1 mg/L	≤1 mg/L	≤1 mg/L
NOEC	<1 mg/L	<1 mg/L	<1 mg/L

EC₅₀– effective concentration for 50% reduction

95%-CL 95 – confidence limits

LOEC – lowest observed effect concentration

NOEC – no observed effect concentration

n.d. – not determined

Table 3 Specific Growth Rate (µ. d^-1)

Concentration		24 h	48 h	72 h
Control	1	2.251	1.693	1.700
	2	2.054	1.591	1.575
	3	2.177	1.639	1.630
	4	2.009	1.619	1.646
	5	2.097	1.727	1.686
	6	1.862	1.632	1.633
	Mean	2.075	1.650	1.645
	SD	0.1356	0.0501	0.0445
	CV	6.5	3.0	2.7
T1 - 1 mg/L	1	1.963	1.464	1.506
	2	1.625	1.490	1.533
	3	1.690	1.540	1.561
	Mean	1.759	1.498	1.533
	SD	0.1792	0.0385	0.0273
	CV	10.2	2.6	1.8
T2 – 3.2 mg/L	1	1.690	1.385	1.418
	2	1.751	1.396	1.462
	3	1.690	1.436	1.456
	Mean	1.710	1.405	1.445
	SD	0.0351	0.0267	0.0238
	CV	2.1	1.9	1.6
T3 – 10 mg/L	1	1.556	1.251	1.304
	2	1.690	1.265	1.321
	3	1.751	1.279	1.332
	Mean	1.665	1.265	1.319
	SD	0.0998	0.0135	0.0143
	CV	6.0	1.1	1.1
T4 – 32 mg/L	1	1.751	0.981	0.957
	2	1.690	0.981	1.005
	3	1.625	1.005	1.021
	Mean	1.688	0.989	0.994
	SD	0.0628	0.0135	0.0333
	CV	3.7	1.4	3.3
T5 – 100 mg/L	1	1.690	0.778	0.603
	2	1.751	0.657	0.542
	3	1.625	0.700	0.563
	Mean	1.688	0.712	0.569
	SD	0.0628	0.0614	0.0309
	CV	3.7	8.6	5.4

Note: Data calculated by ToxRatPro© (Version 3.2.1)

                       SD – standard deviation

                       CV – coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance Butyl-NENA showed significant effects in Pseudokirchneriella subcapitata growth, with an EC50 of 52 mg/L after 72 hours exposure.

Executive summary:

The inhibitory effect of Butyl-NENA on the growth of the freshwater microalgaePseudokirchneriella subcapitata, strain NIVA CHL 1, was investigated. The test was performed according to OECD 201 (2011): Freshwater Alga and Cyanobacteria, Growth Inhibition Test (1).

 

A series of solutions were prepared by dissolving different concentrations of the test substance Butyl-NENA (1 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L) in ISO media 8692, plus respective blanks and a control. The solutions were inoculated with approximately 5Î10⁶cells/L of an exponentially growing culture ofPseudokirchneriella subcapitata. Three replicates of each concentration were incubated in 25 ml glass flasks with 15 ml test volume, in an incubator with orbital shaking set to 22 °C and under continuous light. Six replicate cultures in growth medium were used as controls.Exposure solutions without algal inoculum – blanks – were also made, with six replicates for the control and three replicates for each tested concentration.

 

Growth and growth inhibition were quantified from measurements of fluorescence as a function of time, recorded at 24, 48 and 72 hours and compared with control values. Fluorescence, a surrogate parameter of cell density, was used to avoid exposure to the test operator. A conversion factor between fluorescence and cell counts was determined.The fluorescence values of each blank were measured to verify if there was any interference of the tested substance on the fluorescence readings, whichshowed no interference.

 

A significant inhibition of growth was observed after 72 hours exposure to the test substance Butyl-NENA:

 

 

72 h	Concentration of test substance
EC50	52 mg/L
EC5095%-CL lower	24 mg/L
EC5095%-CL upper	78 mg/L
LOEC	≤1 mg/L
NOEC	<1 mg/L

EC₅₀– effective concentration for 50% reduction

95%-CL 95 – confidence limits

LOEC – lowest observed effect concentration

NOEC – no observed effect concentration