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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:
From 22 May to 07 June 2018
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:
yes
Remarks:
no impact on the study validity
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
no impact on the study validity
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored cold at 4°C in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). A 23-hour stirring period followed by a 1-hour standing period was considered sufficient to ensure equilibration between the test item and aqueous phase.
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 100 mg/L loading rate WAF test group
- Sampling method: not available
- Sample storage conditions before analysis: not available
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).
- Eluate: not specified
- Differential loading: In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L. Based on the results of the range-finding test a "limit test" was conducted at a loading rate of 100 mg/L.
- Controls: control group: reconstituted water (Elendt M7), positive control: potassium dichromate at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not applicable
- 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: not specified
- Justification for species other than prescribed by test guideline: not applicable
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): not available
- Feeding during test: no
- Food type: not applicable
- Amount: not applicable
- Frequency: not applicable

ACCLIMATION
- Acclimation period: not applicable
- Acclimation conditions (same as test or not): not applicable
- Type and amount of food: not applicable
- Feeding frequency: not applicable
- Health during acclimation (any mortality observed): not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: none

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
21-22°C
pH:
8.0-8.1
Dissolved oxygen:
>=3 mg/L
Nominal and measured concentrations:
Range finding test: 1.0, 10 and 100 mg/L nominal loading rate
Definitive test: 100 mg/L nominal loading rate
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Material, size, headspace, fill volume: glass, 150 mL
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): none
- Biomass loading rate: not available

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (Elendt M7)
- Total organic carbon: 21.8 mg C/L at 0 hours
- Particulate matter: none
- Metals: not available
- Pesticides: not available
- Chlorine: not available
- Alkalinity: 7.9 ± 0.3
- Ca/Mg ratio: not available
- Conductivity: not available
- Salinity: not available
- Culture medium different from test medium: not applicable
- Intervals of water quality measurement: not applicable

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20-minute dawn and dusk transition periods.
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L nominal loading rate WAF
- Results used to determine the conditions for the definitive study: cumulative immobilization and sub-lethal effects
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
other: NOEL
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: One Daphnia exhibited reduced mobility at 24 hours but this was considered not to be of biological significance. No sub-lethal effect of exposure was observed throughout the test in the 100 mg/L loading rate WAF
- Observations on body length and weight: not applicable
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: NOEC 0.56 mg/L at 24 and 48 hours, LOEC 1 mg/L at 24 and 48 hours
- Limit test: not applicable
- Dose-response test: not available
- ECx: EC50 0.79 at 24 and 48 hours
- Other: none

Reported statistics and error estimates:
The 48-Hour EL50 value was determined by visual inspection of the data. All statistical analysis for the positive control were carried out using the ToxRat Professional computer software package.

See attached additional information on results.

Validity criteria fulfilled:
yes
Conclusions:
In an acute immobilization test performed on Daphnia magna, and conducted according to OECD test guideline 202 and to GLP, the NOEL value was determined at 100 mg/L loading rate as a Water Accommodated Fraction (WAF) after 24 and 48 hours of exposure to Alfalfa, ext. The effective loading causing 50% response (EL50) was was determined at >100 mg/L loading rate WAF.
Executive summary:

The acute immobilization test conducted according to OECD test guideline 202 and to GLP was performed to assess the acute toxicity of Alfalfa, ext. in Daphnids.

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to a WAF of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation were performed at 0 and 48 hours. A measured concentration of 21.8 mg C/L was obtained at 0 hours whilst 14.7 mg C/L was obtained at 48 hours.

There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours and no sub-lethal effects were observed during the study period.

The NOEL after 24 and 48 hours of exposure was determined at 100 mg/L loading rate WAF. In addition, the effective loading causing 50% response (EL50) was determined at >100 mg/L loading rate WAF.

Description of key information

Daphnia magna (24 and 48 hours):

NOEL: 100 mg/L loading rate WAF

EL50: >100 mg/L loading rate WAF

Key value for chemical safety assessment

Additional information

The acute immobilization test conducted according to OECD test guideline 202 and to GLP was performed to assess the acute toxicity of Alfalfa, ext. in Daphnids.

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to a WAF of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation were performed at 0 and 48 hours. A measured concentration of 21.8 mg C/L was obtained at 0 hours whilst 14.7 mg C/L was obtained at 48 hours.

There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours and no sub-lethal effects were observed during the study period.

The NOEL after 24 and 48 hours of exposure was determined at 100 mg/L loading rate WAF. In addition, the effective loading causing 50% response (EL50) was determined at >100 mg/L loading rate WAF.