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EC number: 205-181-1 | CAS number: 135-16-0
- 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:
- 2018-06-06 to 2018-09-27
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- 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 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
- Version / remarks:
- 2011
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Version / remarks:
- 2008
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid
- EC Number:
- 205-181-1
- EC Name:
- N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid
- Cas Number:
- 135-16-0
- Molecular formula:
- C19H23N7O6
- IUPAC Name:
- (2S)-2-[(4-{[(2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)methyl]amino}phenyl)formamido]pentanedioic acid
- Test material form:
- solid
Constituent 1
Sampling and analysis
- Analytical monitoring:
- yes
- Details on sampling:
- - Concentrations: 0.00 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
- Sampling method: All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 h) and at the end of the exposure (72 h) with algae. The test item and the main degradation product were analysed. The samples were analysed with an LC-MS method. The method was implemented under non-GLP but documented in the raw data and validated according to Annex I. The method validation is not part of this GLP study.
- Sample storage conditions before analysis: Due to the rapid degradation of the test item, the concentrations of the main metabolite of the test item were analytically verified via LC-Q-TOF at the start (0 h) and at the end of the exposure (72 h) in all concentration levels and in the control.
Test solutions
- Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg test item/L were freshly prepared with dilution water. The dispersion was treated with ultrasound for 5 min at room temperature. Thereafter, the dispersion was stirred for 1 h at room temperature at 1100 rpm.
Test organisms
- Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Common name: green fresh water alga
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A three days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 h per day.
ACCLIMATION
- Culturing media and conditions (same as test or not): yes
Study design
- Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
Test conditions
- Hardness:
- The medium has a nominal hardness of 0.24 mmol Ca+Mg/L
- Test temperature:
- Nominal range: 21 - 24 °C, controlled at ± 2 °C
- pH:
- Between 7.27 at start of exposure to 9.50 at the end of exposure
- Nominal and measured concentrations:
- Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
Measured: 0.804 - 2.52 - 8.18 - 23.8 - 79.4 mg/L - Details on test conditions:
- TEST SYSTEM
- Test vessel:Erlenmeyer flasks
- Type (delete if not applicable): closed, with cotton wool plugs
- Material, size, headspace, fill volume: Sterile Erlenmeyer flasks with a fill volume of 250 mL, volume of test medium 100 mL
- Aeration: No
- Initial cells density: Nominal: approximately 5 x E+03 - E+04 cells/mL , Current: 5523 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
GROWTH MEDIUM
- Standard medium used: yes
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4- 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 E-03
Na2MoO4 x 2 H2O 7 E-03
COCI2 x 6 H2O 1.5E-03
CuCl2 x 2 H2O 1E-05
NaHCO3 50
pH 8.1 ± 0.2
- Culture medium different from test medium: No
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Chlorophyll-a-fuorescence and microscopic evaluation
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: 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. Self-fluorescence was observed in the range finding test. Therefore, a separate replicate of each test item concentration was prepared without algae and treated like the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.
- Other: Microscopic evaluation of the cells at the start and the end of exposure was carried out. 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.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Five concentrations were tested in a geometrical series with a (nominal) dilution factor of √10
- Range finding study
- Test concentrations: 12.0 and 120 mg/L
- Results used to determine the conditions for the definitive study: Due to the marked inhibition of growth and yield the test concentrations were chosen below 120 mg/L. - Reference substance (positive control):
- yes
- Remarks:
- potassium dichromate
Results and discussion
Effect concentrationsopen allclose all
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 11.6 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 21.5 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 2.52 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 7.28 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 11.3 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 2.52 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EL10
- Effect conc.:
- 14.6 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EL50
- Effect conc.:
- 28.3 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- other: NOEL
- Effect conc.:
- 3.16 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EL10
- Effect conc.:
- 8.82 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EL50
- Effect conc.:
- 14.2 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- other: NOEL
- Effect conc.:
- 3.16 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Results with reference substance (positive control):
- - Results with reference substance valid? Yes, validity criteria: growth: (Valid Range (average ± 3 x SD)) 0.752 ± 0.554; yield: (Valid Range (average ± 3 x SD)) 0.399 ± 0.294.
- EC50: growth: 0.574 (CI: 0.549 - 0.596), yield: 0.337 (CI: 0.313 - 0.345) - Reported statistics and error estimates:
- EC/EL-values and statistical analyses: EC/EL10-, EC/EL20- and EC/EL,so-values with confidence intervals of growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response.
NOEC/NOEL, LOEC/NOEL and statistical analyses: The NOEC/NOEL and LOEC/NOEL were determined by calculation of statistically significant differences of growth rates and yield. The following statistical tests were conducted: Shapiro-Wilk’s test on normal distribution was calculated with a significance level of 0.01. Levene’s test on variance homogeneity was calculated with a significance level of 0.01. Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm was done for growth rate with a significance level of 0.05.
Applicant's summary and conclusion
- Validity criteria fulfilled:
- yes
- Conclusions:
- In this study, Tetrahydrofolic acid was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values based on calculated concentrations of the test item Tetrahydrofolic acid, measured as the degradation product: The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 21.5 (21.3 - 21.7) mg/L and 11.3 (10.4 - 13.1) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 h were 2.52 mg/L.
Based on the nominal loadings of the test item Tetrahydrofolic acid, the EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 h were 28.3 (28.1 – 28.6) mg/L and 14.2 (12.9 – 16.6) mg/L, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 h were 3.16 mg/L.
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