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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:
5 September 2018 (Study Initiation) to 27 February 2019 (Study Completion)
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No.of test material: MNST9H122A
- 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 as supplied 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: Insoluble in reconstituted water, acetone, DMSO and DMF. During method evaluation, the test item was less soluble in test medium, therefore testing was performed using Water Accommodation Fraction prepared at nominal concentrations, referred to as the loading rate.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: The pH ranged between 7.41 (0 h) and 7.86 (72 h).
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Based on the results of the preliminary range finding study, the main study was performed as a limit study using water accommodated fractions prepared at a loading rate of 100.0 mg/L along with a concurrent control.

- Sampling method: 10 mL of test samples from each replicates were drawn and mixed together for each group at 0 and 48 h. The samples were divided into two equal portions. One portion (20 mL) was sent for test concentration analysis and the second portion (20 mL) was stored at -20 ± 5 ºC temperature till the study completion. Active ingredient concentration in test media was determined using the validated analytical method
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A quantity of 100.1 (≅ 100) mg Fatty acids, C16-18 (even numbered), manganese(II) salts were mixed with reconstituted water and transferred into a 1000 mL volumetric flask and the volume was made up with reconstituted water to obtain a test concentration of 100 mg/L (Stock A). These stocks were kept under continuous (magnetic) stirring at 1000 rpm for 96 h at room temperature. After stirring, test solutions were allowed to re-equilibrate for approximately 1 h. After 1 h,
600 mL of stock solution were collected from the lower portion using an “L” shaped glass tube without disturbing the solution and used for treatment. Prior to adding test solution to vessels, test vessels were pre-conditioned with the respective test concentrations to saturate the surface of the respective vessel to prevent loss of test concentration due to absorption into test vessel walls. Volumes of 99.5 mL from stock A were taken and 0.5 mL daphnia culture added in glass beakers of 600 mL capacity to obtain loading rate of 100.0 mg/L.

- Eluate: reconstituted water
- Differential loading: 0.0 (control), 100.0 mg Fatty acids, C16-18 (even numbered), manganese(II) salts/L.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no, prepared as a Water Accommodated Fraction (WAF)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone:
- Age at study initiation (mean and range, SD): < 24h
- Stage and instar at study initiation: second instar daphnids (less than 24 h old) 2nd instar
- Method of breeding: Gravid females were acclimatised to the 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 was used for the study.
- Source: MicroBio Test Inc, Kleimoer 15, 9030 Mariakerke (Gent), Belgium
- Age of parental stock (mean and range, SD): Not specified
- Feeding during test ; no, Daphnia were not fed during the exposure period
- Food type: live algal cells (Pseudokirchneriella subcapitata)
- Amount: Not specified
- Frequency: Not specified

ACCLIMATION
- Acclimation period: Gravid females were acclimatised to the test conditions for a minimum period of 48 h.
- Acclimation conditions (same as test or not): yes
- Type and amount of food: live algal cells (Pseudokirchneriella subcapitata)
- Feeding frequency: Not specified
- Health during acclimation (any mortality observed): No mortality observed

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
187.2 mg/L CaCO3
Test temperature:
temperature (range: 20.0 to 20.4 °C)
pH:
pH (range: 7.41 to 7.86),
Dissolved oxygen:
dissolved oxygen (range: 7.99 to 8.72 mg/L)
Nominal and measured concentrations:
Nominal: 0 (Control) and 100.0 mg Fatty acids, C16-18 (even numbered), manganese(II) salts/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beaker of 600 mL capacity
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 ml
- Volume of solution: 100 ml, i.e. volumes of 99.5 mL (stock) were taken and 0.5 mL daphnia culture added in glass beakers of 600 mL capacity to obtain loading rate of 100.0 mg/L.
- Aeration: yes
- No. of organisms per vessel: 5 Daphnids
- No. of vessels per concentration (replicates): Four replicates of 5 daphnids per replicate
- No. of vessels per control (replicates): Four replicates of 5 daphnids per replicate
- No. of vessels per vehicle control (replicates): n/a
- Biomass loading rate: 100.0 mg Fatty acids, C16-18 (even numbered), manganese(II) salts/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water was used as the test media, prepared in accordence with OECD 201.All water quality parameters were found to be within specified limits as per OECD guidelines

- Intervals of water quality measurement: Not specified, perpared prior to the study

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A photoperiod of 16 h light and 8 h darkness cycle was maintained with an automatic timer attached to the water bath. Lighting was provided via fluorescent tubes attached on the lid of the low temperature water bath.
- Light intensity: Range: 1310 to 1350 Lux), measured daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The study was conducted as a limit study at a loading rate of 100.0 mg Fatty acids, C16-18 (even numbered), manganese(II) salts/L, no immobility was observed up to 48 h. Therefore, the 24 and 48 h, EL50 (median effective loading concentration for immobilisation) and EC50 (median effective concentration for immobilisation) could not be calculated. Only the EL50 and EC50 were calculated and considered to be EL50 (24 and 48 h), NOELR and LOELR: Greater than 100.0 mg/L
EC50 (24 and 48 h), NOEC and LOEC: Greater than 6.53 mg a.i./L.

VEHICLE CONTROL PERFORMED: /no

RANGE-FINDING STUDY
- Test concentrations: A preliminary range finding study was conducted with 60 daphnids, divided into six groups of 10 daphnids per group. The loading rate concentrations selected for the preliminary range finding study were 0.0 [control], 1.0, 10.0, 25.0, 50.0 and 100.0 mg Fatty acids, C16-18 (even numbered), manganese(II) salts/L.
- Results used to determine the conditions for the definitive study: The percent immobility observed in the dose range finding study were 0, 0, 0, 0, 0 and 0 at loading rates of 0.0 [control], 1.0, 10.0, 25.0, 50.0 and 100.0 mg Fatty acids, C16-18 (even numbered), manganese(II) salts/L, respectively.
Based on the results of the preliminary range finding study, the main study was performed as a limit study using water accommodated fractions prepared at a loading rate of 100.0 mg/L along with a concurrent control.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
6.53 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: none

- - Other biological observations:
- 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: not relavent, study performed as a Water Accommodated Fraction
- Effect concentrations exceeding solubility of substance in test medium: no
Validity criteria fulfilled:
yes
Conclusions:
The EL50 (24 and 48 h), NOELR and LOELR was determines as greater than 100.0 mg/L
The EC50 (24 and 48 h), NOEC and LOEC was determined to be greater than 6.53 mg a.i./L.

Description of key information

The EC50 is considered to be 6.53 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
6.53 mg/L

Additional information