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EC number: 628-907-2 | CAS number: 26661-13-2
- 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
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:
- Experimental Starting Date: 20 November 2012. Experimental Completion Date: 20 December 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
- Deviations:
- yes
- Remarks:
- Please see details on test solutions section
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.2 (Acute Toxicity for Daphnia)
- Deviations:
- yes
- Remarks:
- Please see details on test solution section
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- N4-Benzoylcytosine
- EC Number:
- 628-907-2
- Cas Number:
- 26661-13-2
- Molecular formula:
- C11H9N3O2
- IUPAC Name:
- N4-Benzoylcytosine
Constituent 1
- Specific details on test material used for the study:
- Identification: N-Benzoyl Cytosine
Description: off white powder
Batch: NBC-5-11001
Purity: not supplied
Expiry / retest date: not supplied
Storage conditions: room temperature in the dark over silica gel
Sampling and analysis
- Analytical monitoring:
- yes
- Details on sampling:
- Verification of Test Concentrations
Water samples were taken from the control and the 100% v/v saturated solution test group
(replicates Ri - R4 pooled) at 0 and 48 hours for quantitative analysis. Samples were stored at
approximately -20 °C prior to analysis.
Duplicate samples were taken and stored at approximately -20 °C for further analysis if
necessary.
Test solutions
- Vehicle:
- no
- Details on test solutions:
- In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water
soluble test items, a modification of the standard method for the preparation of aqueous media
was performed. An approach endorsed by several important regulatory authorities in the EU and
elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of
the test item in cases where the test item is of high purity and is poorly soluble in water and in
the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was
prepared by stirring an excess (50 mg/L) of test item in reconstituted water for a period of
24 hours prior to removing any undissolved test item present by filtration (0.2 pm Gelman
Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter) to give a
saturated solution of the test item.
Preliminary solubility work conducted indicated that the test item was practically insoluble in
water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as
defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances
and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to
determine the solubility of the test item under test conditions.
Saturated Solution Preparation
An amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse
osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either
24 or 48 hours. After stirring, samples were taken for chemical analysis after the following pretreatments:
Centrifugation at 10000 g for 30 minutes
Centrifugation at 40000 g for 30 minutes
Filtration through a 0.2 pm Gelman Acrocap filter (approximately 100 mL discarded in
order to pre-condition the filter)
Filtration through a 0.2 pm Gelman Acrocap filter (approximately 500 mL discarded in
order to pre-condition the filter)
Based on this information the test item was prepared using a saturated solution method of
preparation at an initial loading rate of 50 mg/ L, stirred via propeller stirrer for 24 hours prior to
the removal of any undissolved test item by filtration through a 0.2 pm Gelman Acrocap filter
(first approximate 100 mL discarded in order to pre-condition the filter).
Test organisms
- Test organisms (species):
- Daphnia magna
- Details on test organisms:
- The test was carried out using 1st instar Daphnia magna derived from in-house laboratory
cultures.
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium
in a temperature controlled room at approximately 20 °C. The lighting cycle was
controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk
transition periods. Each culture was fed daily with a mixture of algal suspension ( Desmodesmus
subspicatus ) and Tetramin® flake food suspension. Culture conditions ensured that reproduction
was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such
that the young daphnids produced overnight were less than 24 hours old. These young were
removed from the cultures and used for testing. The diet and diluent water are considered not to
contain any contaminant that would affect the integrity or outcome of the study.
Study design
- Test type:
- static
- Water media type:
- freshwater
- Limit test:
- yes
- Total exposure duration:
- 48 h
Test conditions
- Hardness:
- The reconstituted water had an approximate theoretical total hardness of 250 mg/L as
CaC03. - Test temperature:
- 20 - 22°C
the temperature was measured using a Hanna Instruments HI 93510 digital thermometer - pH:
- 7.6 - 8.1
The pH and was measured using a Hach HQ30d Flexi handheld meter - Dissolved oxygen:
- 8.5 - 8.8 mg O2/L
The dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter - Salinity:
- Not applicable as freshwater study
- Conductivity:
- <5 µS cm-1
- Nominal and measured concentrations:
- Range fiinding: 0.10, 1.0, 10 and 100% v/v saturated solution
Definitive test: 100% v/v saturated solution - Details on test conditions:
- Procedure
Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range finding
test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations
of 0.10, 1.0, 10 and 100% v/v saturated solution.
An amount of test item (1125 mg) was added to 22.5 liters of reconstituted water and stirred
using a propeller stirrer for 24 hours at approximately 1500 rpm. After stirring, any undissolved
test item was removed by filtration through a 0.2 pm Gelman Acrocap filter (initial 500 mL
discarded) to give the 100% v/v saturated solution. Serial dilutions were prepared from the
100% v/v saturated solution to give the remainder of the test concentrations.
Each stock solution and prepared concentration was inverted several times to ensure adequate
mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained
in a temperature controlled room at 20 °C to 21 °C with a photoperiod of 16 hours light and
8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each
250 mL test and control vessel contained 200 mL of test media and was covered to reduce
evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to
determine the stability of the test item under test conditions. All samples were stored at
approximately -20 °C prior to analysis. Only concentrations within the range to be used for the
definitive test were analyzed.
Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of
100% v/v saturated solution to confirm that at the highest attainable test concentration, no
immobilization or adverse reactions to exposure were observed.
Experimental Preparation
For the purpose of the definitive test the test item was prepared as a saturated solution.
An amount of test item (1125 mg) was added to 22.5 liters of reconstituted water and stirred
using a propeller stirrer at approximately 1500 rpm for 24 hours. After stirring, any undissolved
test item was removed by filtration through a 0.2 pm Gelman Acrocap filter (initial 500 mL
discarded) to give the 100% v/v saturated solution test concentration.
The concentration and stability of the test item in the test preparations were verified by chemical
analysis at 0 and 48 hours.
Exposure Conditions
In the definitive test 250 mL glass jars containing approximately 200 mL of test preparation were
used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in
the test preparations. Four replicate test and control vessels were prepared. The test vessels
were then covered to reduce evaporation and maintained in a temperature controlled room at
21 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk
transition periods with a light intensity ranging from 647 to 703 lux. The daphnids were not
individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period. Any immobilization or
adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The
criterion of effect used was that Daphnia were considered to be immobilized if they were unable
to swim for approximately 15 seconds after gentle agitation.
Physico-Chemical Measurements
Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen
concentrations and pH were recorded at the start and termination of the test. The pH and
dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter
whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
Results and discussion
Effect concentrationsopen allclose all
- Key result
- Duration:
- 24 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 1.6 mg/L
- Nominal / measured:
- meas. (not specified)
- Conc. based on:
- test mat.
- Basis for effect:
- mobility
- Key result
- Duration:
- 48 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 1.6 mg/L
- Nominal / measured:
- meas. (not specified)
- Conc. based on:
- test mat.
- Basis for effect:
- mobility
- Details on results:
- Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and
100% v/v saturated solution.
Based on this information, a single test concentration of four replicates, of 100% v/v saturated
solution was selected for the definitive test. This experimental design conforms to a "Limit test"
to confirm that at the highest attainable test concentration, no immobilization or adverse
reactions to exposure were observed.
Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 48 hours
showed that measured concentrations of 1.66 and 1.55 mg/L were obtained
respectively indicating that the test item was stable under test conditions.
The analytical methodology was not validated prior to the analysis of range-finding samples; full
validation was later conducted specifically at the concentration employed in the definitive test.
Definitive Test
Verification of Test Concentrations
Analysis of the 100% v/v saturated solution test concentration at 0 and 48 hours
showed that measured concentrations of 1.60 and 1.67 mg/L were obtained respectively.
Immobilization Data
There was no immobilization in 20 daphnids exposed to a test concentration of 100% v/v
saturated solution for a period of 48 hours.
The No Observed Effect Concentration after 24 and 48 hours exposure was 1.6 mg/L.
Observations on Test Item Solubility
Throughout the duration of the test, the test media was observed to be a clear colorless solution.
Physico-Chemical Measurements
Temperature was maintained at approximately 21 °C throughout the test, while there were no treatment related
differences for oxygen concentration or pH.
The oxygen concentration in some of the test vessels was observed to have an air saturation
value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic
air bubbles in the media super-saturating the diluent and was considered not to have had an
impact on the outcome or integrity of the test as no adverse effects were observed in the control
group. - Results with reference substance (positive control):
- A positive control used potassium dichromate as the
reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L
respectively. The No Observed Effect Concentration is based upon zero immobilization at this
concentration.
The results from the positive control with potassium dichromate were within the normal range
for this reference item.
Any other information on results incl. tables
Definitive Test
Inspection of the immobilization data gave the
following results:
Time (h) | EC50 (mg/L) |
24 | >1.6 |
48 | >1.6 |
Positive Control
Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed
Spearman-Karber method (Hamilton et al 1977 ) at 48 hours based on the nominal test
concentrations gave the following results:
Time (h) | EC50 (mg/L) | 95% Confidence limits EC50 (mg/L) |
24 | 0.75 | 0.56 - 1.0 |
48 | 0.45 | 0.42 - 0.48 |
Applicant's summary and conclusion
- Validity criteria fulfilled:
- yes
- Conclusions:
- The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been
investigated and gave a 48-Hour EC50 of greater than 1.6 mg/L based on the mean measured
concentration. The No Observed Effect Concentration was 1.6 mg/L. - Executive summary:
Introduction
A study was performed to assess the acute toxicity of the test item to Daphnia magna. The
method followed was designed to be compatible with the OECD Guidelines for Testing of
Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as
Method C.2 of Commission Regulation (EC) No. 440/2008.
Methods
Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were
exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution
for 48 hours at a temperature of approximately 21 °C under static test conditions. The test item
solution was prepared by stirring an excess (50 mg/L) of test item in test medium using a
propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any
undissolved test item was removed by filtration (0.2 pm Gelman Acrocap filter, first
approximate 500 mL discarded in order to pre-condition the filter) to produce a
100% v/v saturated solution of the test. Immobilization and any adverse reactions to exposure
were recorded after 24 and 48 hours.
Results
Analysis of the 100% v/v saturated solution test concentration at 0 and 48 hours showed that
measured concentrations of 1.60 and 1.67 mg/L were obtained respectively.
The 48-Hour EC50 for the test item to Daphnia magna based on the mean measured test
concentration was greater than 1.6 mg/L. The No Observed Effect Concentration was 1.6 mg/L.
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