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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-08-22 to 2007-11-30
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Sampling method: Water samples were taken from each test chamber prior to test initiation (pre-test), at test initiation and at test termination. Water samples (10 mL) were collected using a 10 mL volumetric pipette and placed in a 20-mL glass scintillation vial .
Details on test solutions:

- Method: The nominal test concentrations were selected on the basis of the reported solubility of the substance (34 μg/L). A primary stock was prepared by isotropic dilution of the 14C-labeled L3 with non-radiolabeled L3. A 7.0 μL aliquot of 14C-L3 (0.00583 g) was added to a 1.0 mL vial. A 200 μL aliquot (0.16344 g) of L3 was also added to the vial. Approximately 0.5 mL of dimethyl formamide (DMF) was added to the vial and the contents of the vial was transferred to a 500-mL volumetric flask. The flask was then brought to volume with DMF, and mixed well. Three 0.1 mL aliquots of the primary stock were counted by LSC. The specific activity of the primary stock was determined to be 6.743 μCi/mg.

Four additional stock solutions were prepared at concentrations of 170, 85, 43 and 21 mg/L by proportional dilution of the primary stock with DMF. DMF only was used as the solvent control. The stock solutions were delivered to the diluter mixing chambers (at a rate of 0.0150 mL/minute) where they were diluted with dilution water (at a rate of 150 mL/min) to achieve the desired test concentrations. Test vessels and mixing chambers remained clear and colourless throughout the test. The concentration of DMF in the solvent control and the treated media was 0.1 mL/L.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:

- Common name: Rainbow trout

- Source: Thomas Fish Company, Anderson, CA., USA

- Length at study initiation (length definition, mean, range and SD): 45 (40-49) mm

- Weight at study initiation (mean and range, SD): 0.73 (0.50-0.88) g

- Feeding during test: None


- Acclimation period: 7 days

- Acclimation conditions: Same as test

- Type and amount of food: Finfish starter #1, Zeigler brothers, Gardners, PA., USA

- Health during acclimation (any mortality observed): No mortality
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
137 mg/L as CaCO3
Test temperature:
Dissolved oxygen:
≥8.6 mg/L (≥82% ASV)
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control), 2.1, 4.3, 8.5, 17 and 34 μg/L

Mean measured concentrations:
The measured concentrations in the treated media corresponded to 57, 49, 55, 52 and 56% of the nominal values respectively.

The results are reported with reference to the mean measured concentrations.
Details on test conditions:
- Exposure vessel type:  25-L polyethylene aquaria containing 15-L of test solution and covered with clear plexiglass lids.

- Delivery of test media: Syringe pumps were used to deliver working stock solutions and solvent to the mixing chambers where they were mixed with dilution water. Dilution water was delivered to the chambers using calibrated rotameters. After mixing the test solutions were split into two replicate test chambers. The test substance is relatively volatile (4 mm at 25°C) and flow rates were adjusted to achieve approximately 7.2 volume additions per day with the aim of maximising exposures. The diluter system was operated for approximately 8 hours before the start of the fish exposures to allow stabilisation.

- Number of replicates, fish per replicate:  Two replicates per treatment, 10 Fish per replicate

- Dilution water source:  Dechlorinated Municipal Water from Bay City, Michigan

- Dilution water chemistry:  Hardness 137 mg/L as CaCO3, Alkalinity 45 mg/L as CaCO3, Conductivity 392  μmhos/cm        

- Vehicle/solvent and concentrations:  The solvent control and treatment groups contained 0.1 mL/L DMF

- Water chemistry in test:  DO, pH and temperature measured in each test chamber daily
Reference substance (positive control):
96 h
Dose descriptor:
Effect conc.:
> 19 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
96 h
Dose descriptor:
Effect conc.:
>= 19 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: none

- Mortality of control: none
Reported statistics and error estimates:
No significant effects on mortality were recorded in the highest treatment. An LC50 value could therefore not be determined.
Sublethal observations / clinical signs:

Table 1. Test results

 Nominal concentration (μg/L)  Mean measured concentration (μg/L)  Mean percentage mortality after 96 hours 
 0 (Control)  <LoQ  0
 0 (Vehicle control)  <LoQ  0
 2.1  1.2  0
 4.3  2.1  0
 8.5  4.7  0
 17  8.9  0
 34  19  0
Validity criteria fulfilled:
A 96-hour LC50 value of >19 μg/L and NOEC of ≥19 μg/L have been determined for the effects of the substance on mortality of Oncorhynchus mykiss based on mean measured concentrations. The results were obtained under flow-through test conditions.

Description of key information

Short-term toxicity to fish: 96-h LC50 >19 μg/L (measured -arith. mean; highest concentration tested) (OECD 203).

Key value for chemical safety assessment

Additional information

A 96-hour LC50 value of >19 μg/L and NOEC of ≥19 μg/L (measured) (highest concentration tested) have been determined for the effects of the substance on mortality of Oncorhynchus mykiss based on mean measured concentrations. In view of the use of flow-through test conditions it is likely that the test organisms were exposed primarily to the parent substance.