Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 November 2010 - 12 November 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Concentration of the test material in the test solutions was determined at the beginning and at the end of the study. Five replicate samples were taken from the test solution. One sample was taken from both control solutions at the start of the test.

Test solutions

Vehicle:
yes
Details on test solutions:
The test item is very poorly soluble in water, furthermore, due to its specific physical character there is no appropriate method for this test system to prepare a saturated test solution (limit test concentration) in aquatic media.
In order to obtain a fine homogeneous suspension which is in the analytically measurable range, acetone as organic solvent was used during the formulation procedure. An amount of 250 mg test item was dissolved in 50 mL acetone thereafter 2×2 mL from this stock solution was diluted in 2×20 L of aquarium water to give the test concentration of 0.5 mg/L (nominal) for two replicates.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Source: HOITSY & RIEGER Kft., Trout Farm, Miskolc-Lillafüred, Hungary
Justification of species: The Rainbow trout (Oncorhynchus mykiss) is one of the convenient species for acute fish toxicity test.
Number of animals: seven fish per test group
Body length of animals: 5.9 – 7.1 cm
Food and Feeding: The fish were not fed during the test
Acclimatisation: >12 days
Animal health: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions, so the fish were healthy. There was no mortality of the population for seven days before the test.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None

Test conditions

Hardness:
The hardness of the dilution water was determined as 225 mg/L (as CaCO3).
Test temperature:
The test temperature was between 15.0 and 15.4 °C.
pH:
The pH was in the range of 8.07 – 8.24.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 93 – 96 % of the air saturation value at the temperature used.
Salinity:
Not reported.
Nominal and measured concentrations:
0.5 mg/L nominal (represented a higher level than the limit of solubility)
0.2 mg/L measured
Details on test conditions:
Equipment and test vessels
Normal laboratory equipment and the following were used for determination of the parameters of the test:
- pH meter
- thermometer
- oxygen meter
- balance
- apparatus for temperature control
- aquaria with total capacity of 25 litres

Environmental test conditions
The water temperature, oxygen concentration and pH were measured at the start and at the end of each renewal period in each test aquarium. The light-dark cycle during the test was 16 hours light and 8 hours darkness.

Description of test procedure
The test duration was 96 hours. Eight fish divided into two replicates (two aquaria per group) were used in test concentration and at each control group (untreated and solvent). A volume of 20 L of test media was used per aquarium. The animals were not fed during the test. The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment. The experiment was carried out under semi-static conditions. The frequency of the water renewal periods was 24 hours. The choice of the test concentration was done on the basis of the results of the preliminary range-finding test.

Preliminary Range-Finding Test
Three fish were exposed to a single test concentration at 0.5 mg/L concentration (nominal) of the test item plus controls, for 96 hours. The test concentration was prepared using acetone as organic solvent as described above. No mortality or any toxic effect was detected in the treated concentration of 0.5 mg/L and in the control groups during the 96 h exposure period.

Concentration Levels Investigated in the Main Test
Because a significant toxic response was not observed during the preliminary concentration range-finding test, only one test concentration of 0.5 mg/L (nominal), one untreated and one solvent control were tested in a limit test. The test results are based on the calculated test item concentrations.

Observations
The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality and any sub-lethal effect were determined in each observation period.
The test conditions (pH, temperature, oxygen saturation) were examined at the start and at the end of each renewal period.
The body weights of the fish were recorded on the day 0 (at the start of the main study). The body weight of 4 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (g fish/litre testing liquid).

Animal welfare
After the experiment, all fish were humanely killed. Euthanasia for all animals was applied by an overdose of an aqueous solution of 100 mg/L Tricaine Methanesulphonate (MS 222; CAS No.: 886-86-2). This technique provides gentle and easy death by direct depression of the brain and vital centers.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Validity
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Concentrations of the test item
The test substance was not detected in the control samples (neither normal nor solvent control). A nominal concentration of 0.5 mg/L was tested in the experiment. The measured concentration was 0.200 mg/L at the start and 0.187 mg/L at the end of the test. The corresponding calculated geometric mean concentration was 0.2 mg/L.

Mortality data
Cumulative mortality data from the exposure of rainbow trout to the test material during the definitive test are given below. There were no mortalities observed in the control and treated groups for a period of 96 hours.

Cumulative mortality data in the definitive test:

Test Group Mortality rate of treated fish (Dead fish/Treated fish)
3h 6h 24h 48h 72h 96h
Control 0/8 0/8 0/8 0/8 0/8 0/8
Solvent Control 0/8 0/8 0/8 0/8 0/8 0/8
0.5 mg/L (nominal) 0/8 0/8 0/8 0/8 0/8 0/8

Sub-lethal effects
No sub-lethal effects were observed in the control and treated groups for a period of 96 hours.

Body weight
The body weight of 4 fish was weighed at the start of the test. There was no considerable difference observed concerning body weights between the groups.
Results with reference substance (positive control):
No positive control used.
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects. The LC50, NOEC and LOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See overall remarks
Conclusions:
The acute toxicity of the test material was assessed with an acute fish toxicity test on rainbow trout (Oncorhynchus mykiss), over an exposure period of 96 hours in a semi-static system.
Under the conditions of this acute fish toxicity study on rainbow trout (Oncorhynchus mykiss), no toxic effect was observed at the tested concentration level. As the concentration of 0.5 mg/L (nominal) represented a higher level than the limit of solubility, it can be stated that the test material had no toxic effect at saturation.
The observed endpoints for the effect of the test material were the following:
The 24h, 48h, 72h and 96h LC50 value: >0.2 mg/L
The 96h NOEC value: 0.2 mg/L
The 96h LOEC value: >0.2 mg/L
The 96h LC100 value: >0.2 mg/L
The results above were based on the measured concentration and determined directly from the raw data.
Executive summary:

In an acute toxicity to fish study (10/095-009H) according to EU Test Method C.1, Oncorhynchus mykiss were exposed to the test material for 96 hours under semi-static conditions.

No toxic effect was observed at the tested concentration level. As the concentration of 0.5 mg/L (nominal) represented a higher level than the limit of solubility, it can be stated that the test material had no toxic effect at saturation.

The observed endpoints for the effect of the test material were the following:

The 24h, 48h, 72h and 96h LC50 value: >0.2 mg/L

The 96h NOEC value: 0.2 mg/L

The 96h LOEC value: >0.2 mg/L

The 96h LC100 value: >0.2 mg/L

The results above were based on the measured concentration and determined directly from the raw data.