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EC number: 226-122-6 | CAS number: 5285-60-9
- 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
Link to relevant study record(s)
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 Jun 2018 - 27 Jul 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
- Version / remarks:
- 2006; Annex 5 corrected 2011
- Deviations:
- no
- GLP compliance:
- yes
- Analytical monitoring:
- yes
- Details on sampling:
- - Concentrations: all test concentrations and the control
- Sampling method: 2.0 mL from the approximate centre of the test vessels at t=0 h, t=24 h and t=72 h
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. - Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. The obtained mixture was allowed to settle for a period of three hours. Thereafter, the 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 the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: Test medium without test item or other additives. - Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum at test initiation: 3 days
- 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.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1 medium, according to NPR 6505
- Pre-culture medium and test medium: M2 medium, according to OECD 201
- Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
- Hardness:
- 24 mg CaCO3/L
- Test temperature:
- 22 - 23°C
- pH:
- At t=0 h: 8.2
At t=72 h: 8.1 - 8.3 - Nominal and measured concentrations:
- Nominal concentrations: 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L
Measured concentrations: 0.000096, 0.00098, 0.022, 0.13, 0.50 mg/L (Time Weighted Average concentrations, see 'Any other information on results' for details on measured concentrations) - Details on test conditions:
- TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 10^4 cells/mL
- Control end cells density: 145 x 10^4 cells/mL
- Replicates: 3 replicates of each test concentration, 6 replicates of the control, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae
GROWTH MEDIUM
- Standard medium used: yes, M2
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for all test concentrations and the control. Temperature of medium: continuously in a temperature control vessel.
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 96 to 98 µE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the 10% SS test group and the control to observe for any abnormal appearance of the algae.
TEST CONCENTRATIONS
- Combined Limit/Range finding study test concentrations: 1.0, 10, 100 % of SS prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate (performed Jul 2018)
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 187 µg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% confidence interval: 183-192 ug/L
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 81 µg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% confidence interval: 78-85 ug/L
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 22 µg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- growth rate
- Remarks on result:
- other: Based on biological relevance
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.98 µg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- growth rate
- Remarks on result:
- other: Based on statistical significance
- Details on results:
- - Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 22 µg/L when compared to the control.
- Growth rates were in the range of the control at the two lowest test concentrations, while inhibition of growth rate increased with increasing concentration from 22 µg/L upwards, resulting in 99% inhibition at the highest concentration. Statistically significant inhibition of growth rates was found at the three highest test concentrations. However, growth rate inhibition was considered to be biologically not relevant at 22 µg/L, where the observed inhibition was below 10%.
- Analytical results: The concentrations measured at the start of the test were 0.0060, 0.022, 0.082, 0.25 and 0.76 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations decreased significantly and were below 30% relative to the initial concentrations at the end of the test in the two highest test concentrations. In the three lowest test concentrations, no concentrations could be detected anymore 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.
- It should be noted that a concentration of 0.055 mg/L was originally measured in the control at the start of the test, while no concentrations could be detected in the samples of the control taken after 24 and 72 hours of exposure. No reason could be identified for this and it was decided to analyse the corresponding reserve sample to investigate whether the sample was contaminated before or after sampling. The concentration detected in the reserve sample was at the level of the carry-over effect observed during the analysis of the samples. As such, it was concluded that the control was not contaminated and that the originally determined concentration was caused by contamination during sampling or preparation of the sample for analysis.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test. - Results with reference substance (positive control):
- - Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ErC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 - 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range. - Reported statistics and error estimates:
- - Statistical significance: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
- ECx calculation: probit analysis using linear max. likelihood regression.
- For the determination of ECx-values for growth rate inhibition, the test group exposed to the lowest test concentration, was excluded from the model fitting procedure. Including the lowest test group interfered with identifying an appropriate non-linear or linear regression model for the calculation of ECx-values. Since no inhibition of growth rate was observed at the two lowest test concentrations the exclusion of the lowest test group was considered to not have impacted the determined ECx-values.
- ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis. - Validity criteria fulfilled:
- yes
- Remarks:
- See 'Overall remarks' for details on validity criteria.
- Conclusions:
- The 72h-EC50 for growth rate inhibition (ErC50) was 187 µg/L (95% confidence interval: 183 - 192 µg/L), based on Time Weighted Average concentrations. The 72h-ErC10 was 81 µg/L (95% confidence interval: 78 - 85 µg/L). The 72h-NOEC was 0.98 µg/L based on statistical significance and 22 µg/L based on biological relevance.
- Executive summary:
In a 72h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to saturated solutions of the test substance prepared at a loading rate of 100 mg/L (1.0, 3.2, 10, 32, 100 % of SS, 3 replicates each) and an untreated control (6 replicates). Measured concentrations of test substance decreased during the exposure period, therefore Time Weighted Average concentrations were calculated and effect parameters were based on TWA concentrations. The 72h-EC50 for growth rate inhibition (ErC50) was 187 µg/L with a 95% confidence interval ranging from 183 to 192 µg/L, based on Time Weighted Average concentrations. The 72h-ErC10 was 81 µg/L (95% confidence interval: 78 - 85 µg/L). The 72h-NOEC for growth rate inhibition was 0.98 µg/L based on statistical significance and 22 µg/L based on biological relevance. The study met all validity criteria, and is considered reliable without restriction.
Reference
Table 1: Time Weighted Average Concentrations
4,4’-methylenebis-N-sec-butylaniline %SS prep. at 100 mg/L |
Measured concentration (mg/L) |
TWA (mg/L) |
||
t=0h |
t=24h |
t=72 h |
||
1.0 |
0.00604 |
0.000012 (b) |
0.000012 (b) |
0.000096 |
3.2 |
0.0224 |
0.00035 |
0.000012 (b) |
0.00098 |
10 |
0.0817 |
0.0513 |
0.000012 (b) |
0.022 |
10 (a) |
0.0777 |
0.0294 |
0.000012 (b) |
0.016 |
32 |
0.248 |
0.197 |
0.041 (c) |
0.13 |
100 |
0.762 |
0.710 |
0.206 |
0.50 |
(a) Without algae.; (b) Estimated as half of the LOD (LOD = 0.023 µg/L); (c) Average of two analyses with different dilutions of the sample.
Table 3: Growth Rate and Percentage Inhibition for the Total Test
Period
4,4’-methylenebis-N-sec-butylaniline |
Mean |
Std. Dev. |
n |
%Inhibition |
Control |
1.659 |
0.0227 |
6 |
|
0.096 |
1.663 |
0.0318 |
3 |
-0.26 |
0.98 |
1.697 |
0.0127 |
3 |
-2.3 |
22 |
1.504 |
0.0361 |
3 |
9.4# |
134 |
1.231 |
0.0408 |
3 |
26 * |
500 |
0.009 |
0.0148 |
3 |
99 * |
* Effect was statistically significant.;#Effect was statistically significant but biologically not relevant (<10%).
Description of key information
72h-ErC50 (growth rate): 187 ug/L (time weighted average concentration)
72h-ErC10 (growth rate): 81 ug/L (time weighted average concentration)
Key value for chemical safety assessment
- EC50 for freshwater algae:
- 187 µg/L
- EC10 or NOEC for freshwater algae:
- 81 µg/L
Additional information
In a 72h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to saturated solutions of the test substance prepared at a loading rate of 100 mg/L (1.0, 3.2, 10, 32, 100 % of SS, 3 replicates each) and an untreated control (6 replicates). Measured concentrations of test substance decreased during the exposure period, therefore Time Weighted Average concentrations were calculated and effect parameters were based on TWA concentrations. The 72h-EC50 for growth rate inhibition (ErC50) was 187 µg/L with a 95% confidence interval ranging from 183 to 192 µg/L, based on Time Weighted Average concentrations. The 72h-ErC10 was 81 µg/L (95% confidence interval: 78 - 85 µg/L). The 72h-NOEC for growth rate inhibition was 0.98 µg/L based on statistical significance and 22 µg/L based on biological relevance. The study met all validity criteria, and is considered reliable without restriction.
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