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EC number: 680-413-6 | CAS number: 217437-44-0
- 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:
- 10 Aug - 19 Sep 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Referenceopen allclose all
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Version / remarks:
- 23 Mar 2006
Annex 5 corrected 28 Jul 2011
- Qualifier:
- according to guideline
- Guideline:
- other: OECD series 23, Guidance document on aquatic toxicity testing of difficult substances and mixtures
- Version / remarks:
- 2000
- GLP compliance:
- yes
Test material
- Reference substance name:
- 2-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]ethyl 2-methylprop-2-enoate
- EC Number:
- 680-413-6
- Cas Number:
- 217437-44-0
- Molecular formula:
- C12H17N3O3
- IUPAC Name:
- 2-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]ethyl 2-methylprop-2-enoate
Constituent 1
Sampling and analysis
- Analytical monitoring:
- yes
- Remarks:
- UPLC
- Details on sampling:
- - Concentrations: A test concentrations and the control at 0, 24, and 72 h
- Sampling method: 1.2 mL from the approximate centre of the test vessels. Reserve samples of 1.2 mL were additionally taken from all test solutions.
- Sample storage conditions before analysis: In a freezer (≤ -15 °C) for a maximum of 3 months.
Test solutions
- Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The preparation of test solutions started with a concentration of 100 mg/L and a 3-d period of magnetic stirring. The obtained mixture was then allowed to settle for a period of 3 h. Then, tha aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of this SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Test solution: After preparation, 50 mL were added to each replicate of the respective test concentration and 1 mL algal suspension was added at a final cell density of 1E+04 cells/mL.
- Differential loading: No
- Controls: Test medium without test item
- Evidence of undissolved material: The test item was a colourless to light yellow liquid and not completely soluble in test medium at the concentration initially prepared. No correction was made for the purity/composition of the test item. All test solutions were clear and colorless at the end of the preparation procedure.
Test organisms
- Test organisms (species):
- other: Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum: 3 d before the start of the test, cells from the algal stock culture were inoculated in M2 pre-culture medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as the test.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 - 24 °C and a light intensity of 60 to 120 µE/m²/s (400 - 700 nm).
Study design
- Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
Test conditions
- Hardness:
- 0.24 mmol/L (Ca+Mg) = 24 mg CaCO3/L (test medium)
- Test temperature:
- 22 °C
- pH:
- 8.2 - 8.3 (0 h)
8.1 - 8.4 (72 h) - Nominal and measured concentrations:
- Control, 1.0, 3.2, 10, 32 and 100% of the saturated solution prepared at a loading rate of 100 mg/L
Control, 0.95, 3.1, 9.5, 32 and 97 mg/L (measured at t = 0 and 72 h) - Details on test conditions:
- TEST SYSTEM
- Test vessel: 100 mL all-glass test vessels with perforated aluminium caps filled with 50 mL test solution
- Initial cell density: 1.0 E+04 cells/mL
- Control end cell density: 230.70 E+04 cells/mL (mean of six replicates)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: The test vessels were randomly distributed in the incubator and repositioned daily. During incubation the algal cells were kept in suspension by continuous shaking.
GROWTH MEDIUM
- Standard medium used: Yes, M1 (according to NPR 6505 "Netherlandse Praktijk Richtlijn no. 6505") and and M2 (according to OECD guideline 201)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium (test medium) was prepared according to OECD guideline 201 using Milli-RO water (tap water purified by reverse osmosis).
- Culture medium different from test medium: Culture medium same as test medium (M2).
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. Temperature was measured continuously in a temperature control vessel.
OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination using TLD-lamps
- Light intensity and quality: 85 - 86 µE*m^-2*s^-1
EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: 24 h intervals by spectrophotometry (680 nm, immersion probe with a path length of 10 mm)
- Appearance of cells: At the end of the test (72 h) by microscope
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx. 3.2
- Range finding study: Yes, a combined limit/range-finding test was performed before the final test
- Test concentrations: 1.0, 3.2, 10, 32 and 100% of the saturated solution prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, a concentration-related increase of algal growth inhibition was observed at all concentrations tested, resulting in 99 and 100% inhibition of growth rate and yield, respectively. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
Results and discussion
Effect concentrationsopen allclose all
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 27 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% confidence interval: 25 - 30 mg/L
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 64 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% confidence interval: 61 - 68 mg/L
- Details on results:
- - Exponential growth in the control: Yes
- Results with reference substance (positive control):
- - Results with reference substance valid? Yes, the obtained effect concentration lies within the historical ranges for growth rate inhibition.
- ErC50 (72 h) = 0.90 mg/L with a 95% confidence interval from 0.88 - 0.93 mg/L
- Other: The reference test was conducted from 02 - 05 Jul 2018 according to OECD guideline 201. - Reported statistics and error estimates:
- Statistical analyses were performed with ToxRat Professional v3.2.1 (ToxRat Solutions GmbH, Germany). The NOEC and ECx values were determined according to the OECD guideline 201 recommendations. Treatment vs control data were statistically compared by the one-side (smaller) Step-down Jonckheere-Terpstra Test procedure (growth rate) or the one-sided (smaller) Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (yield inhibition). The calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test item.
Any other information on results incl. tables
VALIDITY CRITERIA
The study fulfilled the validity criteria defined by the guideline and is thus considered valid and reliable.
Table 1: Validity criteria for OECD 201.
Criterion from the guideline |
Outcome |
Validity criterion fulfilled |
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period. |
In the control, cell density increased by an average factor of 231 within the exposure period. |
Yes |
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35% |
The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 19%. |
Yes |
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. |
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.41%. |
Yes |
ANALYTICAL RESULTS
The measured test item concentrations at the start of the test were 0.95, 3.1, 9.5, 32 and 97 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the saturated solution prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 86 – 99% relative to the initial concentrations at the end of the test. The concentrations measured in the samples without algae were comparable to the concentrations measured in the samples with algae (Table 2).
A small concentration was measured in the control at the end of the test. It was concluded that contamination may have occurred during sampling. The measured value was below the measured value of the lowest calibration standard, with a contribution of 2.0% to the lowest test concentration. Consequently, the response was considered to be negligible and without consequences for the integrity of the present study.
Since all samples taken during the test were at 81 – 101% relative to the nominal concentrations, it was concluded that the undissolved material removed during the preparation of the test samples was not test item related. Thus, the effect concentrations were based on the analytically confirmed nominal concentrations.
Table 2. Analytical results.
Time of sampling
|
Percentage of saturated solution* |
Analyzed concentration |
Relative to initial |
[h] |
[%] |
[mg/L] |
[%] |
0 |
0 |
n.d. |
|
1.0 |
0.948 |
|
|
3.2 |
3.12 |
|
|
10 |
9.47 |
|
|
101 |
9.63 |
|
|
32 |
31.5 |
|
|
100 |
97.4 |
|
|
24 |
0 |
n.d. |
n.a. |
1.0 |
0.958 |
101 |
|
3.2 |
3.19 |
102 |
|
10 |
9.62 |
102 |
|
101 |
9.78 |
102 |
|
32 |
31.5 |
100 |
|
100 |
101 |
103 |
|
72 |
0 |
2 |
n.a. |
1.0 |
0.811 |
86 |
|
3.2 |
2.78 |
89 |
|
10 |
9.01 |
95 |
|
101 |
8.83 |
92 |
|
32 |
30.6 |
97 |
|
100 |
96.5 |
99 |
* Saturated solution prepared at a loading rate of 100 mg/L.
1 Without algae
2 Estimated value of 0.018 mg/L, calculated by extrapolation of the calibration curve (lowest calibration point 0.1 mg/L). Contamination might have occurred during sampling. The maximum contribution to the lowest test concentration, based on measured value and dilution factor, was 2.0%.
n.d. Not detected
n.a. Not applicable
BIOLOGICAL RESULTS
Inhibition of growth rates increased with increasing test item concentrations from 1.0 mg/L upwards, resulting in 75% inhibition at 100 mg/L. Statistically significant inhibition of growth rates was found at all test concentrations (Table 3 and Table 4). However, growth rate inhibition was not considered biologically relevant at 10 mg/L and lower, where the observed inhibition was < 10%. Thus, the NOEC based on biological relevance was set at 10 mg/L.
Inhibition of yield increased with increasing test item concentrations from 1.0 mg/L upwards, resulting in 99% inhibition at 100 mg/L (Table 5). Statistically significant inhibition of yield was found at all test concentrations.
The obtained effect values are summarized in Table 6.
Table 3. Growth rate inhibition.
Nominal test item concentration [mg/L] |
Mean |
S.D. |
n |
% inhibition |
Control |
1.814 |
0.0073 |
6 |
|
1.0 |
1.775 |
0.0066 |
3 |
2.1# |
3.2 |
1.761 |
0.0065 |
3 |
2.9# |
10 |
1.697 |
0.0106 |
3 |
6.4# |
32 |
1.551 |
0.0453 |
3 |
14* |
100 |
0.459 |
0.0986 |
3 |
75* |
* Effect was statistically significant.
# Effect was statistically significant but not biologically relevant (< 10%)
Table 4. Growth rate inhibition at different time intervals.
Nominal test item concentration [mg/L] |
n |
0 – 24 h |
24 – 48 h |
48 – 72 h |
|||
mean |
% inhibition |
mean |
% inhibition |
mean |
% inhibition |
||
Control |
6 |
2.183 |
|
1.771 |
|
1.486 |
|
1.0 |
3 |
2.237 |
-2.4 |
1.603 |
9.5 |
1.487 |
-0.057 |
3.2 |
3 |
2.142 |
1.9 |
1.672 |
5.6 |
1.470 |
1.1 |
10 |
3 |
2.022 |
7.4 |
1.613 |
8.9 |
1.455 |
2.1 |
32 |
3 |
1.890 |
13 |
1.396 |
21 |
1.368 |
8.0 |
100 |
3 |
1.439 |
34 |
0.011 |
99 |
-0.073 |
105 |
Table 5. Yield inhibition.
Nominal test item concentration [mg/L] |
Mean |
S.D. |
n |
% inhibition |
Control |
229.7 |
5.07 |
6 |
|
1.0 |
204.7 |
4.08 |
3 |
11* |
3.2 |
196.2 |
3.88 |
3 |
15* |
10 |
161.4 |
5.20 |
3 |
30* |
32 |
104.6 |
14.78 |
3 |
54* |
100 |
3.1 |
1.23 |
3 |
99* |
* Effect was statistically significant.
Table 6. Summary of effect concentrations (nominal concentrations, analytically confirmed)
Parameter [mg/L] |
NOEC* |
NOEC# |
EC10 |
EC20 |
EC50 |
|
Growth rate |
Value |
< 1.0 |
10 |
27 |
37 |
64 |
|
lower 95%-cl |
|
|
25 |
34 |
61 |
|
upper 95%-cl |
|
|
30 |
39 |
68 |
Yield |
Value |
< 1.0 |
< 1.0 |
3.3 |
6.1 |
20 |
|
lower 95%-cl |
|
|
2.8 |
5.5 |
19 |
|
upper 95%-cl |
|
|
3.8 |
6.8 |
22 |
cI Confidence limit
* Based on statistical significance.
# Based on biological relevance.
Applicant's summary and conclusion
- Validity criteria fulfilled:
- yes
- Remarks:
- For further details please refer to “Any other information on results incl. tables”.
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