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EC number: 279-976-7 | CAS number: 82486-82-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
- Version / remarks:
- 2011
- Deviations:
- no
- 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
- 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
Constituent 1
- 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 concentrationsopen allclose all
- 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
- 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
- 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
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