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EC number: - | CAS number: -
- 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:
- 22 Jul - 14 Sep 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2023
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
- Version / remarks:
- 2006, corrected 28 July 2011
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate
- Cas Number:
- 18641-58-2
- Molecular formula:
- C69H132O8
- IUPAC Name:
- 2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate
1
Sampling and analysis
- Analytical monitoring:
- yes
- Remarks:
- UPLC-MS/MS
- Details on sampling:
- - Concentrations: Samples were taken after 0h, 24h and 72h from all test concentrations (i.e. test vessels at 1.25, 2.5, 5.0, 10 and 20 μg/L inoculated with algae), from the abiotic control (i.e. test vessel at 5.0 μg/L without algae) and from the blank and the solvent controls. Stock solutions used for the preparation of test solutions were sampled as well. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (set to maintain -20°C) until analysis at the analytical laboratory of the Test Facility.
Test solutions
- Vehicle:
- yes
- Remarks:
- tetrahydrofuran (THF)
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Preliminary testing: A non-GLP saturation test was performed in order to select the most appropriate preparation procedure for test solutions. A loading rate of 100 mg/L was continuously stirred for 3 days and concentrations were measured every 24 hours after filtration using a 0.45 μm syringe filter. The measured concentrations in the samples taken after 24, 48 and 72 hours of magnetic stirring were 0.42, 0.63 and 0.33 μg/L by extrapolation of the calibration curve (i.e. below LOQ = 1 μg/L of analytical method in preliminary testing). Based on the obtained results, it was concluded that preparation of test solutions using solvent stocks was required to enable reliable quantification of the test material during the final test.
- Method: The test item was poorly water soluble under test conditions, as confirmed in preliminary testing. Therefore, to enable reliable preparation of test concentrations and reliable quantification of the test material, the test solutions were prepared using a suitable organic solvent according to the OECD Guidance Document 23. The preparation of the stock solutions for the final test, i.e. test material in tetrahydrofuran (THF), started with the highest concentration of 0.20 mg/mL. The highest concentrated stock solution was vigorously shaken to completely dissolve the test material in THF. The lower stock concentrations, i.e. 0.0125, 0.025, 0.050 and 0.10 mg/mL, were prepared by subsequent dilutions of the highest concentrated stock in THF. Subsequently, a volume of 100 μL of each stock solution was added to respectively 1.0 L test medium to obtain the according test concentrations. The mixtures were magnetically stirred for 15 minutes.
- Controls: Blank control (test medium only) and solvent control (prepared by adding 100 μL THF to 1.0 L test medium)
- Other controls: Abiotic control (i.e. test vessel at 5.0 μg/L without algae) to check maintenance of actual concentrations
- Chemical name of vehicle: Tetrahydrofuran (THF)
- Concentration of vehicle in test medium: 100 μL/L in all test concentrations and in solvent control. Organic solvent concentrations up to 100 μL/L are allowed according to OECD 201.
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.
Test organisms
- Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: The algae were cultured in OECD medium at a temperature of 21-24°C and continuously aerated and exposed to light (light intensity 60 to 120 μE/m2/s in wavelength range of 400 to 700 nm).
- Age of inoculum: A pre-culture was prepared from the algal stock culture 3 days before the start of the test under the same conditions as used in the test.
Study design
- Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
Test conditions
- Test temperature:
- 22 - 23°C
- pH:
- 8.0 - 8.2
- Nominal and measured concentrations:
- Nominal: Controls (blank and solvent), 1.25, 2.5, 5.0, 5.0 (abiotic control without algae), 10 and 20 μg/L
Measured (0h): < LOQ, 1.20, 2.29, 4.90, 5.05, 9.67 and 17.8 μg/L
Measured (24h): < LOQ, < LOQ, < LOQ, < LOQ, < LOQ, 1.10 and 0.876 μg/L
Measured (72h): < LOQ for all controls and all test concentrations
Measured (TWA): < LOQ, 0.21, 0.26, 0.34, 0.35, 1.3 and 1.5 μg/L
LOQ = 0.25 μg/L in the final test - Details on test conditions:
- TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminum caps, perforated for ventilation, containing 50 mL of test solution.
- Initial cells density: 1x10^4 cells/mL
- Control end cells density: 218x10^4 cells/mL in the blank control and 233x10^4 cells/mL in the solvent control
- No. of vessels per concentration: 3 replicates. Extra replicates (1-2) per concentration were prepared without algae for sampling/effect parameters measurements
- No. of vessels per blank control: 6 replicates
- No. of vessels per vehicle control: 6 replicates
GROWTH MEDIUM
- Standard medium used: Yes
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium M2
- Culture medium different from test medium: OECD medium M1
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity: 79 to 80 μE/m2/s
- Other: 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 (with observation intervals if applicable) :
- 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 = 20 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes, a non-GLP range-finding test was performed at a loading rate of 100 mg/L. The test solution was stirred with a magnetic stirrer for 3 days and then the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 μm membrane filter and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. No inhibition of algal growth rate or yield was found at any of the tested concentrations. An additional GLP range finding test was conducted using 0.25, 1.0 and 4.0 μg/L test concentrations (prepared using THF similarly as in the final test), a blank control and a solvent control. No significant inhibition of algal growth rate or yield was found at any of the test groups in the additional range-finding test. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
Results and discussion
Effect concentrationsopen allclose all
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 1.5 µg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- > 1.5 µg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 1.5 µg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Details on results:
- - Exponential growth in the control: Yes
- Results with reference substance (positive control):
- - Results with reference substance valid? Yes (dating March 2022)
- EC50 (72 h): 0.95 mg/L, 95% confidence limits: 0.87 - 1.0 mg/L - Reported statistics and error estimates:
- ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used to perform the statistical analysis. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Dunnett`s Multiple t-test Procedure, α=0.05, one-sided, smaller). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s Test) and a test for homogeneity of the variances (Levene’s test).
The ECx-values could not be determined because the observed effects were below 10%.
Any other information on results incl. tables
VALIDITY CRITERIA
The study fulfilled the validity criteria defined by the guideline (Table 1) 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. | Actual values: 218 in the blank control and 233 in the solvent control | 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% | Actual values: 26% in the blank control and 10% in the solvent control | 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. | Actual values: 1.3% in the blank control and 2.2% in the solvent control | Yes |
ANALYTICAL RESULTS
The analytical findings are summarized in Table 2.
Table 2. Analytical results
Nominal concentration (μg/L) | Measured concentration (μg/L) at 0h | Measured concentration (μg/L) at 24h | Measured concentration (μg/L) at 72h | TWA (μg/L) |
Controls (blank and solvent) | <LOQ | <LOQ | <LOQ | <LOQ |
1.25 | 1.20 | <LOQ* | <LOQ* | 0.21 |
2.5 | 2.29 | <LOQ* | <LOQ* | 0.26 |
5.0 | 4.90 | <LOQ* | <LOQ* | 0.34 |
5.0** | 5.05 | <LOQ* | <LOQ* | 0.35 |
10 | 9.67 | 1.10 | <LOQ* | 1.3 |
20 | 17.8 | 0.876 | <LOQ* | 1.5 |
*Limit of Quantification (LOQ) = 0.25 μg/L. Therefore, half of the LOQ (i.e. 0.125 μg/L) was used to calculate the time-weighted average exposure concentrations (TWA).
**Abiotic control (without algae)
BIOLOGICAL RESULTS
Biological findings are summarised in Table 3, Table 4, Table 5 and Table 6.
Table 3. Results in control replicates
| Individual Cell Densities [x104cells/mL] | Section-by-Section Growth Rates [day-1] | Growth rates [day-1] | CV growth [%] | Yields [x104cells/mL] | CV yield [%] | |||||
| 0 h | 24 h
| 48 h | 72 h | 0 – 24 h | 24 – 48 h | 48 – 72 h | 72 h | 72 h | 72 h
| 72 h
|
Blank Control | 1.000 | 3.233 | 39.570 | 201.410 | 1.173 | 2.505 | 1.627 | 1.768 | 1.3 | 200.410 | 7.1 |
1.000 | 3.875 | 39.147 | 202.334 | 1.355 | 2.313 | 1.643 | 1.770 | 201.334 | |||
1.000 | 4.963 | 41.661 | 218.730 | 1.602 | 2.128 | 1.658 | 1.796 | 217.730 | |||
1.000 | 4.944 | 46.388 | 211.550 | 1.598 | 2.239 | 1.517 | 1.785 | 210.550 | |||
1.000 | 4.197 | 44.675 | 237.434 | 1.434 | 2.365 | 1.670 | 1.823 | 236.434 | |||
1.000 | 4.349 | 43.755 | 233.746 | 1.470 | 2.309 | 1.676 | 1.818 | 232.746 | |||
Mean | 1.000 | 4.260 | 42.533 | 217.534 | 1.439 | 2.310 | 1.632 | 1.793 | 216.534 | ||
Solvent Control | 1.000 | 6.671 | 40.228 | 214.998 | 1.898 | 1.797 | 1.676 | 1.790 | 2.2 | 213.998 | 12.4 |
1.000 | 6.702 | 37.779 | 196.209 | 1.902 | 1.729 | 1.647 | 1.760 | 195.209 | |||
1.000 | 7.666 | 47.821 | 251.649 | 2.037 | 1.831 | 1.661 | 1.843 | 250.649 | |||
1.000 | 8.690 | 44.859 | 230.742 | 2.162 | 1.641 | 1.638 | 1.814 | 229.742 | |||
1.000 | 6.993 | 40.191 | 225.212 | 1.945 | 1.749 | 1.723 | 1.806 | 224.212 | |||
1.000 | 8.723 | 51.890 | 278.278 | 2.166 | 1.783 | 1.680 | 1.876 | 277.278 | |||
Mean | 1.000 | 7.574 | 43.795 | 232.848 | 2.018 | 1.755 | 1.671 | 1.815 | 231.848 |
Table 4. Results in treatments.
Nominal concentration [μg/L] | Cell Densities [x104cells/mL] | Growth rates [day-1] | CV growth [%] | Yields [x104cells/mL] | CV yield [%] | |||
0 h | 24 h | 48 h | 72 h | 72 h | 72 h | 72 h | 72 h | |
1.25 | 1.000 | 7.378 | 44.159 | 213.352 | 1.788 | 1.1 | 212.352 | 6.2 |
1.000 | 7.308 | 42.711 | 225.828 | 1.807 | 224.828 | |||
1.000 | 9.243 | 46.738 | 241.237 | 1.829 | 240.237 | |||
Mean | 1.000 | 7.976 | 44.536 | 226.806 | 1.808 | 225.806 | ||
2.5 | 1.000 | 6.449 | 37.240 | 199.363 | 1.765 | 2.0 | 198.363 | 11.2 |
1.000 | 7.166 | 39.551 | 197.409 | 1.762 | 196.409 | |||
1.000 | 9.068 | 43.486 | 239.339 | 1.826 | 238.339 | |||
Mean | 1.000 | 7.561 | 40.092 | 212.037 | 1.784 | 211.037 | ||
5.0 | 1.000 | 6.984 | 40.408 | 221.013 | 1.799 | 1.9 | 220.013 | 10.1 |
1.000 | 6.940 | 36.685 | 197.248 | 1.761 | 196.248 | |||
1.000 | 9.029 | 48.391 | 241.314 | 1.829 | 240.314 | |||
Mean | 1.000 | 7.651 | 41.828 | 219.858 | 1.797 | 218.858 | ||
10 | 1.000 | 5.901 | 33.713 | 183.285 | 1.737 | 2.9 | 182.285 | 14.9 |
1.000 | 6.835 | 40.830 | 226.365 | 1.807 | 225.365 | |||
1.000 | 7.547 | 46.367 | 247.137 | 1.837 | 246.137 | |||
Mean | 1.000 | 6.761 | 40.303 | 218.929 | 1.794 | 217.929 | ||
20 | 1.000 | 6.657 | 38.038 | 208.280 | 1.780 | 0.7 | 207.280 | 3.8 |
1.000 | 6.937 | 42.716 | 214.148 | 1.789 | 213.148 | |||
1.000 | 7.178 | 44.906 | 224.248 | 1.804 | 223.248 | |||
Mean | 1.000 | 6.924 | 41.887 | 215.559 | 1.791 | 214.559 |
Table 5. Yields/Growth rates and Percentage Inhibition for the Total Test Period
Nominal concentration [μg/L] | Growth rate (mean) [day-1] | Growth rate inhibition [%] compared to control | Yield (mean) [x104cells/mL] | Yield inhibition [%] compared to control |
72 h | 72 h | 72 h | 72 h | |
Solvent control | 1.815 |
| 231.8 |
|
1.25 | 1.808 | 0.39 | 225.8 | 2.6 |
2.5 | 1.784 | 1.7 | 211.0 | 9.0 |
5.0 | 1.797 | 1.0 | 218.9 | 5.6 |
10 | 1.794 | 1.2 | 217.9 | 6.0 |
20 | 1.791 | 1.3 | 214.6 | 7.5 |
Table 6. Effect values based on Initial Measured Concentrations and Average Exposure Concentrations (TWA)
Effect parameters | Initial Measured Concentrations | Average Exposure Concentrations (TWA) |
NOEC (growth and yield) | ≥ 20 μg/L | ≥ 1.5 μg/L |
ErC10 and EyC10 (72 h) | > 20 μg/L | > 1.5 μg/L |
ErC50 and EyC50 (72 h) | > 20 μg/L | > 1.5 μg/L |
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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