Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 947-842-3 | 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
Link to relevant study record(s)
- Endpoint:
- effects on growth of green algae
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 2022-04-04 to 2022-04-07
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Version / remarks:
- (2016)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Version / remarks:
- (2011)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- Test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide
Batch number LAB AA101.20 Grobgut
Purity (certified) 100% (UVCB)
Chemical composition Disodium 2- (dodecanoylamino)pentanedioate (main component)
Appearance Colorless powder (solid)
Water Solubility 7.8 g/L
Stability under test
conditions Not specified
Expiry date 2023-08-23
Recommended storage Room temperature - Analytical monitoring:
- yes
- Remarks:
- via LC-MS/MS
- Details on sampling:
- Sampling schedule
All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.
Sampling and pre-treatment
At the start, samples were taken from one additional replicate of each test item concentration and the control and at the end samples were taken from pooled replicates. - Vehicle:
- no
- Details on test solutions:
- Stock solution, media preparation
100 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the solution was visually clear. The Tyndall effect was negative.
Preparation of the test item solutions
Out of the stock solution 6 concentrations were tested in a geometric series with a dilution factor of √10: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg test item/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP). - Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- Test organism Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)
Synonyms
Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)
Reason for the selection of the test organism
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.
Origin
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.
Culture medium Nutrient medium Z according to LÜTTGE et al. (1994) - Test type:
- static
- Water media type:
- freshwater
- Remarks:
- Dilution water (OECD TG 201 medium)
- Limit test:
- no
- Total exposure duration:
- 72 h
- Hardness:
- see section "Any other information on results incl. tables" below.
- Test temperature:
- see section "Any other information on results incl. tables" below.
- pH:
- see section "Any other information on results incl. tables" below.
- Nominal and measured concentrations:
- Out of the stock solution 6 concentrations were tested in a geometric series with a dilution factor of √10: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg test item/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).
The measured concentrations of N-Lauroyl-L-glutamic acid in the fresh media were in the range of 86 to 96% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 109 to 120% of the nominal test item concentrations. - Details on test conditions:
- Control
Six replicates were exposed under the same conditions as the test concentrations.
Reference Item
Potassium dichromate was tested as a reference item (for results of the most recent study, please refer to "Any other information on results incl. tables" ).
Test method Static procedure
Duration of the test 72 hours
Replicates Six replicates for the control, three replicates per concentration level
Test container Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
Test volume 100 mL
Ultrapure water Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm).
Preculture
A three days old preculture, prepared in dilution water, was used as inoculum.
Initial cell density
Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 6864 cells/mL
Application
Application was carried out by adding an appropriate amount of algae inoculum to the test solutions.
Incubation The flasks were positioned randomly and repositioned daily.
Temperature
Nominal range: 21 - 24 °C, controlled at ± 2 °C
Agitation
Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
Light intensity (target) Approximately 4440 to 8880 lux
Light regime 24 hours/day light
Light homogeneity Within ± 15% over incubation area
Type and Frequency of Measurements
Biological Parameters
Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the preliminary range finding test at the concentration of 100 mg/L.
Microscopic evaluation
The algae cells were evaluated microscopically at the start and the end of the incubation period. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.
Physico-chemical Properties
The pH-value at the start of the exposure was measured in one additional replicate of each test item concentration and the control. At the end of the exposure, it was measured in a pooled sample of the test item concentrations and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test. - Reference substance (positive control):
- yes
- Remarks:
- potassium dichromate (SIGMA ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9)
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 80.7 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- yield
- Remarks on result:
- other: Confidence Intervals 61.2 - > 100 mg/L
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC20
- Effect conc.:
- 16.9 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 6.41 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 3.16 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 1 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 100 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC20
- Effect conc.:
- > 100 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 51.2 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.:
- 31.6 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.:
- 10 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Details on results:
- Biological Data
Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities for all test item concentration levels and control. All effect values given are based on the nominal test item concentration of fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide.
Physico-chemical Data
The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media were clear by eye throughout exposure period (possible turbidity related to algae growth not taken into account).
Measured Exposure Concentrations during the Definitive Test
The concentrations of N-Lauroyl-L-glutamic acid, component of the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide, and the control were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-MS/MS.
The measured concentrations of N-Lauroyl-L-glutamic acid in the fresh media were in the range of 86 to 96% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 109 to 120% of the nominal test item concentrations. - Results with reference substance (positive control):
- Test of the Reference Item
The toxicity of potassium dichromate (SIGMA ALDRICH, batch number BCCC1619, purity 100.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2021-11-01 to 2021-11-04. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2006, and the limits were set using the threefold standard deviation of these values.
For details see "Any other information on results incl. tables" below. - Reported statistics and error estimates:
- EC-values and statistical analyses
EC10-,EC20- and EC50- values with confidence intervals of growth rate inhibition and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression with the GraphPad Prism Software.
NOEC, LOEC and analyses
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.
The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Monotonicity of Concentration/Response was tested by trend analysis by contrast with a significance level of 0.05 for growth rate.
Step-down Jonckheere-Terpstra Test Procedure was performed with a significance level of 0.05 for growth rate.
Dunnett’s multiple -t-test was performed with a significance level of 0.05 for yield.
Software The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using the following software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.
• ToxRat Version 3.3.0, ToxRat Solutions GmbH - Validity criteria fulfilled:
- yes
- Conclusions:
- In this study, fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations):
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L and 80.7 (61.2 – > 100) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 10.0 and 1.00 mg/L, respectively. - Executive summary:
Aquatic effects of fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide (Batch no.: LAB AA101.20 Grobgut) to the unicellular freshwater green alga Pseudokirchneriella subcapitata were determined at the test facility according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2022-04-04 to 2024-04-07. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.
The study was conducted under static conditions with an initial cell density of 6864 cells/mL. A stock solution with a nominal concentration of 100 mg test item/L was prepared with dilution water (see Table 2). The dispersion was agitated until the solution was clear by eye. The Tyndall effect was negative. Six concentrations were tested in a geometrical series with a dilution factor of √10: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg test item/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.
The concentrations of the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide and the control were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-MS. Details of the analytical method are presented in section 14.
The measured concentrations of N-Lauroyl-L-glutamate in the fresh media were in the range of 86 to 96% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 109 to 120% of the nominal test item concentrations. Therefore, all effect values are given are based on nominal test item concentrations.NOEC, LOEC and ECx-values with 95% Confidence Intervals of fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide (0-72 hours)
based on the nominal test item concentrations[mg/L]Inhibition of Growth Rate NOEC 10.0 LOEC 31.6 ErC10 51.2 (36.6 – 67.6) ErC20 > 100 ErC50 > 100 Inhibition of Yield NOEC 1.00 LOEC 3.16 EyC10 6.41 (0.785 – 13.1) EyC20 16.9 (9.68 – 27.5) EyC50 80.7 (61.2 - > 100)
Reference
Cell Densities
Nominal test item concentration | Replicate | Cell density [cells/mL] | |||
[mg/L] | No. | 0 hours | 24 hours | 48 hours | 72 hours |
100 | 1 | 6864 | 16516 | 79223 | 593052 |
2 | 6864 | 15079 | 85835 | 432091 | |
3 | 6864 | 15493 | 89748 | 749312 | |
Mean | 6864 | 15696 | 84935 | 591485 | |
31.6 | 1 | 6864 | 18513 | 127298 | 920979 |
2 | 6864 | 20603 | 127639 | 1022229 | |
3 | 6864 | 19870 | 123654 | 1048182 | |
Mean | 6864 | 19662 | 126197 | 997130 | |
10.0 | 1 | 6864 | 23819 | 157110 | 1129776 |
2 | 6864 | 24458 | 156454 | 1146823 | |
3 | 6864 | 22948 | 160142 | 1256350 | |
Mean | 6864 | 23742 | 157902 | 1177650 | |
3.16 | 1 | 6864 | 20839 | 161500 | 1190814 |
2 | 6864 | 21696 | 151317 | 1123793 | |
3 | 6864 | 24991 | 153224 | 1279559 | |
Mean | 6864 | 22509 | 155347 | 1198055 | |
1.00 | 1 | 6864 | 28391 | 200498 | 1372893 |
2 | 6864 | 27863 | 205842 | 1363042 | |
3 | 6864 | 21855 | 163048 | 1471264 | |
Mean | 6864 | 26036 | 189796 | 1402400 | |
0.316 | 1 | 6864 | 27321 | 155005 | 1291299 |
2 | 6864 | 22998 | 157371 | 1205207 | |
3 | 6864 | 25301 | 163929 | 1375547 | |
Mean | 6864 | 25207 | 158768 | 1290684 | |
Control | 1 | 6864 | 29932 | 189793 | 1389041 |
2 | 6864 | 28225 | 192096 | 1333940 | |
3 | 6864 | 24635 | 171090 | 1391874 | |
4 | 6864 | 28184 | 185862 | 1322470 | |
5 | 6864 | 28301 | 202549 | 1390750 | |
6 | 6864 | 22170 | 178305 | 1343701 | |
Mean | 6864 | 26908 | 186616 | 1361963 |
Evaluation after 72 hours
Statistically significant differences of growth rates and yield compared to
control values are marked (s), not significant differences are marked (ns).
Nominal test item concentration | Replicate | Growth rate | Inhibition of growth rate | Yield | Inhibition of yield | ||
[mg/L] | No. | [d-1] | [%] | [cells/mL] | [%] | ||
100 | 1 | 1.49 | 16 | 586188 | 57 | ||
2 | 1.38 | 22 | 425227 | 69 | |||
3 | 1.56 | 11 | 742448 | 45 | |||
Mean | (s) | 1.48 | 16 | (s) | 584621 | 57 | |
31.6 | 1 | 1.63 | 7 | 914115 | 33 | ||
2 | 1.67 | 5 | 1015365 | 25 | |||
3 | 1.68 | 5 | 1041318 | 23 | |||
Mean | (s) | 1.66 | 6 | (s) | 990266 | 27 | |
10.0 | 1 | 1.70 | 4 | 1122912 | 17 | ||
2 | 1.71 | 3 | 1139959 | 16 | |||
3 | 1.74 | 2 | 1249486 | 8 | |||
Mean | (s)* | 1.71 | 3 | (s) | 1170786 | 14 | |
3.16 | 1 | 1.72 | 3 | 1183950 | 13 | ||
2 | 1.70 | 4 | 1116929 | 18 | |||
3 | 1.74 | 1 | 1272695 | 6 | |||
Mean | (s)* | 1.72 | 2 | (s) | 1191191 | 12 | |
1.00 | 1 | 1.77 | 0 | 1366029 | -1 | ||
2 | 1.76 | 0 | 1356178 | 0 | |||
3 | 1.79 | -1 | 1464400 | -8 | |||
Mean | (ns) | 1.77 | -1 | (ns) | 1395536 | -3 | |
0.316 | 1 | 1.75 | 1 | 1284435 | 5 | ||
2 | 1.72 | 2 | 1198343 | 12 | |||
3 | 1.77 | 0 | 1368683 | -1 | |||
Mean | (ns) | 1.75 | 1 | (ns) | 1283820 | 5 | |
Control | 1 | 1.77 | 1382177 | ||||
2 | 1.76 | 1327076 | |||||
3 | 1.77 | 1385010 | |||||
4 | 1.75 | 1315606 | |||||
5 | 1.77 | 1383886 | |||||
6 | 1.76 | 1336837 | |||||
Mean | 1.76 | 1355099 |
* = statistical significant, but biologically not relevant (Inhibition < 5%)
Section-by-Section and Average Specific Growth Rates of the Control Group
(0 - 72 hours)
Replicate No. | Specific Growth Rate [d-1] | Mean | SD | CV | Mean CV [%] | |||
section-by-section | (0 - 72 hours) | ± | [%] | |||||
0 - 24 hours | 24 - 48 hours | 48 - 72 hours | ||||||
Control | 1 | 1.47 | 1.85 | 1.99 | 1.77 | 0.267 | 15.1 | 20.0 |
2 | 1.41 | 1.92 | 1.94 | 1.76 | 0.297 | 16.9 | ||
3 | 1.28 | 1.94 | 2.10 | 1.77 | 0.434 | 24.5 | ||
4 | 1.41 | 1.89 | 1.96 | 1.75 | 0.298 | 17.0 | ||
5 | 1.42 | 1.97 | 1.93 | 1.77 | 0.307 | 17.3 | ||
6 | 1.17 | 2.08 | 2.02 | 1.76 | 0.509 | 28.9 | ||
Mean | 1.76 | |||||||
SD ± | 0.01 | |||||||
CV [%] | 0.45 |
SD = Standard deviation CV = Coefficient of variation
Environmental Conditions
Nominal test item concentration | pH-values | ||||||
[mg/L] | Start; 0 hours | End; 72 hours | |||||
100 | 7.67 | 8.74 | |||||
31.6 | 7.61 | 9.00 | |||||
10.0 | 7.55 | 9.24 | |||||
3.16 | 7.54 | 9.28 | |||||
1.00 | 7.58 | 9.26 | |||||
0.316 | 7.58 | 9.29 | |||||
Control | 7.85 | 9.22 | |||||
Room temperature [°C]: | min.: 22.0 | max.: 23.0 | mean value: 22.5 | ||||
n = 19 | mean value: 5853 | CV [%]: 8.09 | |||||
Light intensity | range of the measured values: 5090 - 6586 | ||||||
[lux] | equalling -13.0 to 12.5 |
CV = Coefficient of variation n = number of measuring points
Water parameters of the Dilution Water
Parameters of the dilution water (measured on 2022-03-22) | ||||
Conductivity | Total hardness | Acidity | Alkalinity | Total organic carbon |
[µS/cm] | [mg CaCO3/L] | [mmol/L] | [mmol/L] | [mg C/L] |
137 | 34 | 0.1 | 0.6 | < LOQ* |
*Limit of quantification = 2.00 mg C/L
Measured Concentrations of N-Lauroyl-L-glutamic acid during the Definitive Test
Sampling date | Fresh media, | Old media, | |||
0 hours | 72 hours | ||||
Nominal | N-Lauroyl-L-glutamic acid | ||||
Concentration of | |||||
the test item | the active substance | Meas. conc. | [%] | Meas. conc. | [%] |
[mg/L] | [mg a.s./L] | [mg/L] | [mg/L] | ||
100 | 58.1 | 50.4 | 87 | 68.1 | 117 |
31.6 | 18.4 | 16.4 | 89 | 21.5 | 117 |
10.0 | 5.81 | 5.01 | 86 | 6.93 | 119 |
3.16 | 1.84 | 1.72 | 93 | 2.00 | 109 |
1.00 | 0.581 | 0.527 | 91 | 0.654 | 112 |
0.316 | 0.184 | 0.177 | 96 | 0.221 | 120 |
Control | < LOQ | < LOQ |
Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percentage of the nominal concentration of the test item
LOQ = limit of quantification (0.100 mg test item/L, corresponding to 0.0581 mg a.s./L)
EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study | Valid Range (average ± 3 x SD) | |
Growth Rate inhibition | ||
ErC50 | 1.09 | 0.787 ± 0.566 |
95% confidence interval | 0.944 – 1.25 | |
Yield inhibition | ||
EyC50 | 0.643 | 0.429 ± 0.348 |
95% confidence interval | 0.581 – 0.712 |
SD = Standard deviation
Validity Criteria
Validity Criterion | Required | This study |
Increase of the cell growth in the control cultures | Exponentially, ≥ 16-fold corresponding to a specific growth rate of 0.92 day-1 | 198-fold |
(specific growth rate 1.76 day-1) | ||
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures | ≤ 35% | 20.0% |
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures | ≤ 7% | 0.45% |
Gradient Table
Time [min] | A [%] | B [%] |
0.00 | 70 | 30 |
0.30 | 70 | 30 |
0.80 | 5 | 95 |
2.00 | 5 | 95 |
2.10 | 70 | 30 |
2.50 | 70 | 30 |
Dilution Steps
Nominal | Dilution | Sample | Final |
test item concentration | Factor | volume | volume |
[mg/L] | [mL] | [mL] | |
100 | 20000 | 0.051) | 5.01) |
0.052) | 5.02) | ||
31.6 | 5000 | 0.051) | 5.01) |
0.22) | 5.02) | ||
10.0 | 2000 | 0.051) | 5.01) |
0.12) | 1.02) | ||
3.16 | 500 | 0.051) | 5.01) |
0.42) | 1.02) | ||
1.00 | 200 | 0.051) | 5.01) |
0.316 | 50 | 0.21) | 5.01) |
Control | 20 | 0.11) | 1.01) |
* including factor 2
1) first dilution step 2) second dilution step
Parameter, Acceptance Criteria and Results of the Method Validation
Parameter | Acceptance criteria | Result | |
Calibration | ≥ 5 standard concentrations, | 1.0 to 10 µg standard/L (n = 6), | ü |
r ≥ 0.99 (R2 ≥ 0.9801 for 2nd order regression) | r ≥ 0.99 | ||
Calibration range from ≤ 30% LOQ to 20% above highest concentration level after sample preparation | |||
Matrix effects | Should be ≤ 20% | Matrix effects ≤ 20% | ü |
Lowest calibration level (LCL) | S/N ≥ 9 for quantifier ion trace | S/N for 1.0 µg standard/L | ü |
S/N ≥ 3 for qualifier ion trace | 2262 (Quantifier), 208 (Qualifier) | ||
Limit of Quantification (LOQ) | Should be at or below lowest nominal test concentration | 0.100 mg test item/L (1 x LOQ) | ü |
120 mg test item/L (1200 x LOQ) | |||
Accuracy (Fortified samples) | Mean recovery rate of 70-120% | 1 x LOQ: 99% (n = 5) | ü |
per fortification level (2 levels) | 1200 x LOQ: 94% (n = 5) | ||
Precision | Relative standard deviation ≤ 20% per fortification level | 1 x LOQ: 4.6% | ü |
1200 x LOQ: 9.4% | |||
Stability | Samples: Stable if 70 – 120% of nominal concentration | See section 18.1 | ü |
Calibration standards: Stable if ±10% of freshly prepared standard | |||
Specificity: LC-MS/(MS) | Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity). | quantifier [m/z]: 330.13 > 148.00 | ü |
qualifier [m/z]: 330.13 > 129.98 | |||
Blank values < 30% of the LOQ | Blank values < 30% of LOQ | ü | |
Procedural recovery | Procedural recoveries with experimental samples. The recovery has to be 70-120% of the nominal value. | See section 18.2 | ü |
Preparation of Fortified Samples
LOQ Level | Control | 1 | 1200 |
Stock solution | - | 6000 | 6000 |
[mg test item/L] | (Dilution medium 1) | (Dilution medium 1) | |
(Medium) | |||
Spiking solution | - | 10 | Stock solution |
[mg test item/L] | (Algae dilution water) | ||
(Medium) | |||
Replicates | 2 | 5 | 5 |
Concentration of the LOQ | - | 0.100 | 120 |
[mg test item/L] | |||
Medium for preparation | Algae dilution water | ||
Volume of spiking solution [mL] | - | 0.05 | 0.1 |
Volume of medium [mL] | 5.0 | 4.95 | 4.90 |
Dilution factor | 20 | 20 | 20000 |
Dilution medium | Acetonitrile containing 2% formic acid1) | ||
Dilution medium 22)3) | |||
Sample volume [mL] | 51) | 51) | 51) |
0.12) | 0.12) | 0.12) | |
0.13) | |||
Finale volume [mL] | 101) | 101) | 101) |
1.02) | 1.02) | 102) | |
103) | |||
Final expected concentrations for analyses [µg/L] | - | 5.0 | 6.0 |
Dilution medium 1: Acetonitrile : algae dilution water (50 : 50) containing 1% formic acid
Dilution medium 2: Acetonitrile : ultra-pure water (50 : 50) containing 1% formic acid
1) First dilution step
2) Second dilution step
3) Third dilution step
Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of N-Lauroyl-L-glutamic acid, component of the test item fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide
Fortified concentrations*: 0.101 mg test item/L (1 x LOQ) and 121 mg test item/L (1200 x LOQ), corresponding to 0.0584 mg a.s./L (1 x LOQ) and 70.1 mg a.s./L (1200 x LOQ).
Replicate | N-Lauroyl-L-glutamic acid | |||
1 x LOQ | 1200 x LOQ | |||
Meas. conc. | % | Meas. conc. | % | |
[mg/L] | [mg/L] | |||
1 | 0.0568 | 97 | 75.4 | 108 |
2 | 0.0585 | 100 | 63.9 | 91 |
3 | 0.0620 | 106 | 68.5 | 98 |
4 | 0.0547 | 94 | 59.5 | 85 |
5 | 0.0586 | 100 | 62.6 | 89 |
Mean | 0.0581 | 99 | 66.0 | 94 |
SD ± | 0.003 | 6.2 | ||
CV [%] | 4.6 | 9.4 |
Meas. conc. = Measured concentration of the test item, dilution factor taken into account
% = Percent concentration of the fortified sample
* = Weighing factor taken into account
a.s. = active substance
SD = Standard deviation
CV = Coefficient of Variation
Stability
The stability of the fortified samples was checked after 7 days and was 116% of the nominal concentration. Samples were found to be stable over this period.
The stability of the stock solution (100 mg standard/L in methanol) was checked after 33 days and was 102 and 106% of the nominal concentration. The stock solution was found to be stable over this period.
The responses of a 3 days old calibration standard (6 µg test item/L) was compared with the responses of a freshly prepared calibration standard with the same concentration. The deviation was < 10% and was found to be stable over this period.
Procedural Recovery
A procedural recovery (Quality Control) was freshly prepared on each day of analysis. It was treated in parallel to the test samples. For results, see Table 16.
Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test
Sampling | 0 hours | 72 hours | 72 hours1) | |||
date | ||||||
Quality Control | N-Lauroyl-L-glutamic acid | |||||
Test item | Meas. | % | Meas. | % | Meas. | % |
[mg/L] | conc. | conc. | conc. | |||
[mg a.s./L] | [mg a.s./L] | [mg a.s./L] | ||||
1.00 | 0.0512 | 88 | 0.0629 | 108 | 0.0579 | 99 |
Meas. conc. = measured concentration of the test item, dilution and weighing factors taken into account
% = percent of the nominal concentration
a.s. = active substance
1) = reanalyzed samples
Preliminary Test (non-GLP)
A non-GLP preliminary range finding test under static conditions over a period of 72 hours was conducted at the test facility. A stock solution with a concentration of 100 mg test item/L was freshly prepared with dilution water and agitated until the solution was visually clear. Out of this solution two concentration levels were prepared (by dilution) with the concentrations 1.00 and 10.0 mg/L.
In the range finding test, two replicates per concentration and four for the control were tested.
Results of the Range Finding Test (0 - 72 hours)
Nominal test item concentration | Growth Rate Inhibition | Yield Inhibition |
[mg/L] | [%] | [%] |
100 | 19 | 62 |
10.0 | 2 | 11 |
1.00 | 2 | 9 |
Measured Exposure Concentrations during the non-GLP Preliminary Range Finding Test
Determination of the test item via LC-MS
Sampling | 0 hours | 72 hours | |||
Start of the exposure | End of the exposure | ||||
(with algae) | (with algae) | ||||
Nominal concentration of the | fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide | ||||
test item [mg/L] | active substance | Meas. conc. | % | Meas. conc. | % |
[mg a.s./L] | [mg a.s./L] | [mg a.s./L] | |||
100 | 65.9 | 62.0 | 94 | 77.1 | 117 |
10.0 | 6.59 | 6.00 | 91 | 7.84 | 119 |
1.00 | 0.659 | 0.630 | 96 | 0.758 | 115 |
Control | < LCL | < LCL |
Meas. Conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal test item concentration
a.s. = active substance (N-Lauroyl-L-glutamate, disodium salt)
LCL = lowest calibration level = 0.5 µg test item/L corresponding to 0.330 µg a.s./L
Description of key information
EC50 (72h) > 100 mg/L, EC10 (72h) = 51.2 mg/L (nominal) (OECD 201)
Key value for chemical safety assessment
- EC50 for freshwater algae:
- 100 mg/L
- EC10 or NOEC for freshwater algae:
- 51.2 mg/L
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.