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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:
31 August 2018 (Study Initiation) to 27 March 2019 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities, Pvt. Ltd.
- Lot of test material: IRST7H112A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in original container as received by the Sponsor. Container kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: As the test item was not soluble in any suitable solvent, the test item was mixed with test media and administered as a nominal concentration filtrate.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

Analytical monitoring:
no
Remarks:
As the test item was determined as highly insoluble in water and below the limit of analytical detection, dose formulations were administered as a nominal concentration filtrate without determination of homogeneity, concentration and stability.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: As the test item was determined as highly insoluble in water and below the limit of analytical detection, dose formulations were administered as a nominal concentration filtrate without determination of homogeneity, concentration and stability. As dose formulations were prepared by single dilution and administered within two hours of preparation, this exception does not impact the outcome of the study or the interpretation of the results.
- Controls: Reconstituted water was used as the test media.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, stock solution filtered prior to test system administration.
- Other relevant information: None
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: Not specified
- Justification for species other than prescribed by test guideline: None, as per OECD 202 guideline recomendation
- Age at study initiation (mean and range, SD): Healthy Daphnia magna (showing no signs of stress) less than 24 h old (second instar) cultured and bred in the test facility were used for the study. Second instar daphnids (less than 24 h old) were separated from the adults in a culture of Daphnia.
- Stage and instar at study initiation: Healthy Daphnia magna (showing no signs of stress) less than 24 h old (second instar) cultured and bred in the test facility were used for the study.
- Source: The master culture of Daphnia magna was originally procured from MicroBio Test Inc, Kleimoer 15, 9030 Mariakerke (Gent), Belgium.
- Age of parental stock (mean and range, SD): Not specified
- Feeding during test: Daphnia were not fed during the exposure period.
- Food type: Daphnia were fed with live algal cells (Pseudokirchneriella subcapitata)
- Amount: Not specified
- Frequency: Not specified

ACCLIMATION
- Acclimation period: Acclimatised to test conditions for a minimum period of 48 h
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Daphnia were fed with live algal cells (Pseudokirchneriella subcapitata)
- Feeding frequency: Not specified
- Health during acclimation (any mortality observed): No


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Gravid females were acclimatised to test conditions for a minimum period of 48 h. During this period, Daphnia were fed with live algal cells (Pseudokirchneriella subcapitata). Freshly hatched daphnids belonging to the same stock (less than 24 h-old) were collected using a micropipette and used for the study. Daphnia were not fed during the exposure period.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Standard test item exposure duration for OECD 202
Hardness:
176.8 mg/L as CaCO3
Test temperature:
19.7 ± 0.3°C
pH:
7.41 ± 0.03
Dissolved oxygen:
8.49 ± 0.03 mg/L
Nominal and measured concentrations:
Prelim test (nominal) concentrations: 0.0 (Control), 1.0, 10.0, 50.0, and 100.0 mg Fatty acids, C16-18 (even numbered), iron(III) salts/L
Main test (nominal) concentration: 0.0 (Control), 100.0 mg Fatty acids, C16-18 (even numbered), iron(III) salts/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 600 mL beaker
- Type: open
- Fill volume: 100 mL
- Volume of solution: 100 mL
- Aeration: yes, the diluent water was aerated prior to use so that the dissolved oxygen concentration had reached saturation.
- No. of organisms per vessel: 5 Daphnids
- No. of vessels per concentration (replicates): Each group consisted of four replicates with 5 daphnids per replicate, i.e. 20 daphnids per group.
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: As per OECD 202
- Culture medium different from test medium: no, as per OECD 202
- Intervals of water quality measurement: Water quality parameters, viz., temperature, pH and dissolved oxygen were measured at 0 and 48 h. Water hardness was measured at the beginning of the test only. All water quality parameters were found to be within specified limits as per OECD guidelines.

OTHER TEST CONDITIONS
- Adjustment of pH: 7.48 to 7.89
- Dissolved oxygen (range: 7.48 to 8.91 mg/L)
- Photoperiod: 16h light / 8h dark
- Light intensity: 1340 to 1360 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): EC50 (24 and 48 h), NOEC and LOEC

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Nominal test concentrations: 0.0 (Control), 1.0, 10.0, 50.0, and 100.0 mg Fatty acids, C16-18 (even numbered), iron(III) salts/L
- Results used to determine the conditions for the definitive study:
Immobilisation in the control was 0% (not to exceed 10%) at termination, thus the validity criteria was met.
Dissolved oxygen concentration was ≥ 7.48 mg/L in the control and test vessels at the end of the test, thus the validity criteria was met.
Daphnia in the control groups did not show immobilization, or other signs of disease or stress, e.g., discolouration or unusual behaviour such as trapping at the surface of water.

Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
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
Details on results:
- Behavioural abnormalities:
- Observations on body length and weight: not specified
- Other biological observations: None
- Mortality of control: None
- Other adverse effects control: None
- Immobilisation of control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None,dose formulations administered as a nominal concentration filtrate
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
- Results with reference substance valid? , yes, the validity and reliability of the test system Daphnia magna was confirmed by conducting a positive control study using potassium dichromate.
- Relevant effect levels: yes, the appropriate positive control response was observed for Potassium Dichromate when evaluating % immobilisation at 24 h and 48 h and behavioual symptons, against control groups.
- EC50 at 24 h: 0.59 mg/L
- EC50 at 48 h: 0.40 mg/L
- NOEC: 0.09 mg/L
- LOEC: 0.20 mg/L
Reported statistics and error estimates:
As the study was conducted as a limit test, probit analysis was not carried out.
Validity criteria fulfilled:
yes
Conclusions:
The EC50 (24 and 48 h), NOEC and LOEC were all greater than 100.0 mg Fatty acids, C16-18 (even numbered), iron(III) salts/L (nominal concentration).

Description of key information

The 48h ERC50 was found to be > 100 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information