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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
04 March - 20 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
- Lot No. of test material: 07-170-FIL-2/3
- Appearance: White powder
- Expiration date: May 2010
- Purity: 98.4%
- Storage condition of test material: Ambient
Analytical monitoring:
yes
Details on sampling:
The concentration of test substance was determined in each test solution at 0 and 96 hours by standard analytical methods that were validated prior to dosing (not described in the report).
Vehicle:
yes
Remarks:
Dimethyl formamide (DMF)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was determined to be insoluble in water. Therefore, a water-soluble fraction was prepared by adding the required nominal amount of test substance in moderately hard synthetic freshwater and mixing the solution for 20-24 hours. After this mixing time, the undissolved test substance was separated from the solution and only this water-soluble fraction was used for testing.
- Controls: Untreated control and solvent control
- Chemical name of vehicle: Dimethyl formamide (DMF)
- Concentration of vehicle in test medium: DMF was added as a solvent to the water-soluble fraction at 12 mL per 24 L dilution in water.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Trout Lodge, Sumner, Washington
- Age at study initiation: Juvenile; approx. 3 weeks old for range-finding test; approx. 6 weeks old for definitive test
- Length at study initiation: Range-finding: 26 - 32 mm; Definitive: 35 - 42 mm
- Weight at study initiation: Range-finding: 0.2210 - 0.2932 g; Definitive: 0.4691 - 0.5782 g

ACCLIMATION
- Acclimation period: at least 7 days
- Acclimation conditions (same as test or not): yes
- Type and amount of food during acclimation: Not specified
- Feeding frequency during acclimation: Not specified
- Health during acclimation: No signs of disease, injury, stress or mortality were observed

FEEDING DURING TEST: No - feeding stopped 48 hours before the start of the test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
Range-finding: 95 mg/L CaCO3
Definitive: 90 mg/L CaCO3
Test temperature:
12 ± 2°C (actual temperature in definitive test: 11°C)
pH:
Range-finding: pH 8.5
Definitive: measured pH
- 0 hours: 8.3 - 8.4
- 24 hours: 7.7 - 7.8
- 48 hours: 7.2 - 7.3
- 72 hours: 7.5 - 7.6
- 96 hours: 7.7 - 7.9
Dissolved oxygen:
≥ 60% saturation

Measured dissolved oxygen:
- 0 hours: 10.8 - 11.2 mg/L
- 24 hours: 6.5 - 7.5 mg/L
- 48 hours: 5.8 - 6.4 mg/L
- 72 hours: 5.9 - 6.2 mg/L (treatment groups were aerated)
- 96 hours: 9.8 - 10.2 mg/L
Salinity:
No data
Conductivity:
Definitive test (measured):
- 0 hours: 230 - 240 µmhos/cm
- 24 hours: 225 - 230 µmhos/cm
- 48 hours: 225 - 235 µmhos/cm
- 72 hours: 230 - 235 µmhos/cm
- 96 hours: 230 - 235 µmhos/cm
Nominal and measured concentrations:
Nominal concentrations: 100 and 1000 mg/L
Measured: See table 1
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquarium
- Material, size, headspace, fill volume: 2.5 gallon glass aquaria; filled with 700 mL of the appropriate solution
- Aeration: yes (72 hours after dosing)
- No. of organisms per vessel: 10 organisms per vessel
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: one control group, 3 replicates
- No. of vessels per vehicle control: one solvent control group (12 mL DMF in 24 mL dilution water added to the stock solution), 3 replicates
- Biomass loading rate: Maximum loading 0.8 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Moderately hard laboratory freshwater

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light/ 8 hours dark cycle

EFFECT PARAMETERS MEASURED: At 0, 6, 24, 48, 72 and 96 hours following dosing, each aquarium was examined for mortality and the number of live fish were recorded.

TEST CONCENTRATIONS
- Range finding study: Organisms were randomly placed (10 each) into the aquarium containing the appropriate concentration of the test substance. Ten organisms that were not exposed to the test substance served as controls to demonstrate the condition of the test population. At 0, 6, 24, 48, 72 and 96 hours following dosing, each aquarium was examined for mortality and the number of live fish was recorded.
- Test concentrations: 1, 10, 100, 500 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Mortality was less than 10%.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality: None - a 100% survival rate was observed in treated fish
- Behavioural abnormalities: No data
- Mortality of control: None - a 100% survival rate was observed in control fish
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The water was observed to be cloudy in the 1000 mg/L test group.

Table 1: Dose verification

 

Control

Solvent control DMF

mg/L

Time point

NA

NA

100

1000

Day 0

0.56

0.57

1.18

1.70

96 hour

0.54

0.53

0.62

0.84

 

Validity criteria fulfilled:
yes
Remarks:
Both the range-finding and limit test control mortality were less than 10%.
Conclusions:
The 96 hour LC50 of Formononetin was determined to be >1000 mg/L. The NOEC for survival was 1000 mg/L.

Description of key information

A GLP study was performed in accordance with OECD Guideline 203, to determine the toxicity of the test substance, Formononetin, to Oncorhynchus mykiss in a 96 hour static, non-renewal test. The 96 hour LC50 of Formononetin was determined to be >1000 mg/L and the NOEC for survival was 1000 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
1 000 mg/L

Additional information

The endpoint is not required for Annex VII registrations but an acute fish toxicity study had been performed in 2008 for other regulations and has therefore been submitted as supporting information under REACH.