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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
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 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004-04-13
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008-05-30
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 medium of the only test concentration and 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 concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 48 hours).
All samples were diluted by a factor of 2 with acetonitrile.

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 the only test concentration, and in the duplicate control samples, from both sampling times (0 and 48 hours).
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test item was weighed and dissolved
- Controls: yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): reconstituted water Elendt "M4"
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: clone 5
- Source: ibacon
- Feeding during test: at least on all working days
- Food type: green algae (Desmodesmus subspicatus)
- Frequency: at least on all working days

ACCLIMATION
- Acclimation period: not necessary, since the test was performed in the same medium as the culturing
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3
Alkalinity: 0.9 mmol/L
Test temperature:
20.9 to 21.1 °C at test start
20.1 to 20.2 °C at test end
pH:
7.7 at test start
7.7 to 8.0 at test end
Dissolved oxygen:
8.8 to 9.2 mg/L at test start
7.3 to 8.5 mg/L at test end
Nominal and measured concentrations:
Nominal : 100 mg/L
At the start of the test 119% of the nominal test concentration were found (100 mg test item/L). After 48 hours test duration, 109% of the nominal value was determined (100 mg test item/L). During the test the daphnids were exposed to a mean of 114% of nominal. Therefore, all reported results refer to nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass beakers of 100 mL volume containing approximately 60 mL of test medium. The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates): 4 per treatment group
- No. of vessels per vehicle control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
Analytical grade salts and additives were added at the following nominal concentrations in deionised water (conductivity < 5 µScm-1):

Main compounds:
CaCl2·2 H2O 293.80 mg/L
MgSO4·7 H2O 123.30 mg/L
KCl 5.80 mg/L
NaHCO3 64.80 mg/L
Na2SiO3·9 H2O 10.00 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L

Trace elements:
H3BO3 2860 µg/L
MnCl2·4 H2O 361 µg/L
LiCl 306 µg/L
RbCl 7 1 µg/L
SrCl2·6 H2O 152 µg/L
NaBr 16 µg/L
Na2MoO4·2 H2O 63 µg/L
CuCl2·2 H2O 17 µg/L
ZnCl2 13 µg/L
CoCl2·6 H2O 10 µg/L
KI 3.3 µg/L
Na2SeO3 2.2 µg/L
NH4VO3 0.6 µg/L
Na2EDTA·2 H2O 2.5 mg/L
FeSO4·7 H2O 1.0 mg/L

Vitamins:
Thiamin HCl 75.0 µg/L
Cyanocobalamin (B12) 1.0 µg/L
Biotin (B6) 0.75 µg/L


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 500 - 670 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):
yes
Remarks:
Most recent Test with the Reference Item Potassium dichromate performed in January 2017
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50, EC20, EC10, NOEC, LOEC
- Limit test: no
- Dose-response test: yes
- ECx: EC50 (48 h) = 1.06 mg/L; EC20 (48 h) = 0.967 mg/L; EC10 (48 h) = 0.922 mg/L
- Other: NOEC (48 h) = 0.5 mg/L; LOEC (48 h) = 1.0 mg/L
Reported statistics and error estimates:
Statistical analysis was not necessary.
The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.
Validity criteria fulfilled:
yes
Conclusions:
L-4-Hydroxyproline did not exhbit aquatic toxicity to Dahnia magna at a test concentration of 100 mg/L under the conditions of the test according to OECD 202.
Executive summary:

The toxic effect of the test item L-4-hydroxyproline toDaphnia magna was assessed in a static limit test. The 48-hour NOEC was determined to be 100mg test item/L. The 48-hour LOEC was determined to be > 100mg test item/L and the 48-hour EC50value was determined to be > 100mg 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 119% of the nominal test concentration were found (100 mg test item/L). After 48 hours test duration, 109% of the nominal value was determined (100 mg test item/L). During the test the daphnids were exposed to a mean of 114% of nominal. Therefore, all reported results refer to nominal concentrations.

L-4-Hydroxyproline did not exhbit aquatic toxicity to Dahnia magna at a test concentration of 100 mg/L under the conditions of the test according to OECD 202.

Description of key information

L-4-Hydroxyproline did not exhbit aquatic toxicity to Dahnia magna at a test concentration of 100 mg/L under the conditions of the test according to OECD 202.

This result is supported by a study with tubified worms. The acute toxicity of Cu decreases in the presence of L-4-hydroxyproline. The findings demonstrate that even 20 mM of L-4-hydroxyproline alone are not harmful to tubified worms.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Remarks:
EC50 > 100 mg/L
Effect concentration:
100 mg/L

Additional information