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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-03 to 2017-08-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011-07-28
Qualifier:
according to guideline
Guideline:
other:  Commission Regulation (EC) No 761/2009, Annex, Part C, C.3.: "Freshwater Algae and Cyanobacteria, Growth Inhibition Test", Official Journal of the European Union (EN)
Version / remarks:
2009-08-24
Qualifier:
according to guideline
Guideline:
other:  SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Thai Kyowa Biotechnologies Co., Ltd.
- Expiration date of the lot/batch: 2018-02-22
- Purity test date:2016-03-08

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:At 20°C +/- 5°C, in the dark
- Solubility and stability of the test substance in the solvent/vehicle: highly soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:
Analytical monitoring:
yes
Details on sampling:
The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
The samples were diluted by a factor of two with acetonitrile.
One additional sample of the control and of the dilution solvent was taken at test start and test end without any sample treatment.

All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and were kept stored up to the date of the final report.

The concentrations of the test item L-4-hydroxyproline were analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples, from both sampling times (0 and 72 hours).
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Weighed amounts were dissolved; stock solution was diluted
- Controls: Reconstuted water, potassium dichromate (historical)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): reconstuted water (OECD medium)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s)
including control(s)): stock solution: 100 mg/L; test concentrations were 100, 50, 25, 12.5 and 6.25 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): all test material dissolved
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.4 to 22.8 °C
pH:
8.0 at test start
7.9 to 9.1 at end of test
Nominal and measured concentrations:
mg/L % of nominal RSD
6.25 107 5
12.5 109 4
25 111 6
50 107 9
100 110 3
Number of analysed samples: 4 each
RSD: relative standard deviation
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass
- Initial cells density: approx. 5000/mL
- Control end cells density: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): positive control by historical data
- No. of vessels per vehicle control (replicates): 1 per test concentration

GROWTH MEDIUM
- Standard medium used: yes
TEST MEDIUM / WATER PARAMETERS
Analytical grade salts were added at the following nominal concentrations in deionised water:
Macronutrients:
NaHCO3 50.0 mg/L
CaCl2 x 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 x 7 H2O 15.0 mg/L
MgCl2 x 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace elements:
Na2EDTA x 2 H2O 100.0 μg/L
FeCl3 x 6 H2O 64.0 μg/L
MnCl2 x 4 H2O 415.0 μg/L
H3BO3 185.0 μg/L
Na2MoO4 x 2 H2O 7.0 μg/L
ZnCl2 3.0 μg/L
CoCl2 x 6 H2O 1.5 μg/L
CuCl2 x 2 H2O 0.01 μg/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: Mean light intensity: 7058 lux (range: 6320 to 7470 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 100, 50, 25, 12.5, 6.25 mg/L (nominal)
Reference substance (positive control):
yes
Remarks:
Most recent test with the test item potassium dichromate perforemd in November / December 2016
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 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.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
71.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
45.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
36.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 12.5 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration. Beginning with 25 mg test item/L the cells showed deformations, were smaller and had a more roundish shape compared to the cells of the control.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.12 mg/L (growth rate)
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by a 3 parametric normal concentration distribution function.
For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Welch t-test (yield) and Step-down Jonckheere-Terpstra test (growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH
Validity criteria fulfilled:
yes
Conclusions:
L-4-Hydroxyproline influenced the growth of Pseudokirchneriella subcapitata in high concentration. The impact on the growth rate was comparatively low. The 3 most important effect concentrations were determined to be:
72-hour NOEC: 25 mg/L
72-hour LOEC: 50 mg/L
72-hour EC50: 71.6 mg/L
Executive summary:

The influence of L-4-hydroxyproline on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 39.2 mg test item/L and the 72-hour ErC50 value was calculated to be 71.6 mg test item/L. The 72-hour NOEyC was determined to be 25 mg test item/L and the associated 72-hour LOEyC was 50 mg test item/L. The 72-hour NOErC was determined to be 25 mg test item/L and the associated 72-hour LOErC was 50 mg test item/L.

The quantification of the test item L-4-hydroxyproline in the test samples was performed using liquid chromatography with MS/MS detection.

At the start of the test 105% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 113% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 109% of nominal. Therefore, all reported results refer to nominal concentrations.

L-4-Hydroxyproline influenced the growth of Pseudokirchneriella subcapitata in high concentration. The impact on the growth rate was comparatively low. The 3 most important effect concentrations were determined to be:

72-hour NOEC: 25 mg/L

72-hour LOEC: 50 mg/L

72-hour EC50: 71.6 mg/L

Description of key information

L-4-Hydroxyproline influenced the growth of Pseudokirchneriella subcapitata in high concentration. The impact on the growth rate was comparatively low. The 3 most important effect concentrations were determined to be:

72-hour NOEC: 25 mg/L

72-hour LOEC: 50 mg/L

72-hour EC50: 71.6 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
71.6 mg/L
EC10 or NOEC for freshwater algae:
25 mg/L

Additional information