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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 November 2010 - 11 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 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
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.
Both at the start and at the end of the study four 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 0.1 mL from this stock solution was diluted in 1000 mL of ISO medium to give the test concentration of 0.5 mg/L (nominal).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Source: Supplied by National Institute of Public Health.
Cultured under standardised conditions at the Ecotoxicological Laboratory of LAB Research Ltd..
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 20 animals in test groups and control groups, divided into 4 replicates (5 animals / replicate).
Age of the animals: They were less than 24 h old at the beginning of the test.
Acclimatization: There was no acclimatization because the water used was similar to the culture water.
Food and feeding: Before the test the Daphnia culture was fed with concentrated algal suspension of Pseudokirchneriella subcapitata. The test animals were not fed during the test.

Study design

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

Test conditions

Hardness:
The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
The water temperature was measured at the start and at the end of each renewal period in each test vessel. The test temperature was in the range of 20.9 – 21.1 °C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.7 and 21.4 °C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of each renewal period in each test vessel and was in the range of 8.04 – 8.16.
Dissolved oxygen:
The dissolved oxygen concentration was measured in each test vessel at the start and at the end of each renewal period and was in the range of 6.7 – 6.8 mg/L.
Salinity:
Not reported
Nominal and measured concentrations:
0.5 mg/L nominal (represented a higher level than the limit of solubility)
0.3 mg/L measured
Details on test conditions:
Dilution water
Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.

Equipment
Normal laboratory equipment and the following apparatus were necessary for the determination of test parameters:
- pH meter
- thermometer
- oxygenmeter
- balance
- climate chamber
As test vessels laboratory glass beakers were used (volume: ~50 mL).

Light: The light-dark cycle during the test was 16 hours light and 8 hours darkness.

Description of the test procedure
The test duration was 48 hours. Twenty animals, divided into four groups (glass beaker) of five animals each (at least 4 mL test solution/animal) were used at the test concentrations and for the controls. The animals were not fed during the test. Because the test item was not stable for 48 hours during the preliminary experiments, the definitive test was performed under semi-static conditions. The frequency of the water renewal periods were 24 hours.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours. The test solutions were prepared using acetone as organic solvent as described above.
The concentration levels used and results (48 h) of the preliminary range-finding test are summarised below.

Results of the Preliminary Range-Finding Test

Nominal concentrations [mg/L] Untreated control Solvent control 0.125 0.25 0.5

Number of treated/immobilised animals 10/0 10/0 10/0 10/0 10/0

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 immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
The water temperature, oxygen concentrations and pH of the test solutions and the control were measured at the beginning and at the end of each renewal period.
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
VALIDITY
There were no immobilized animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. 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 0.260 mg/L at the start and 0.280 mg/L at the end of the test. The corresponding calculated geometric mean concentration was 0.3 mg/L.

Immobilisation
The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour.
There was no immobilisation in 20 daphnids exposed in each group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.
Results with reference substance (positive control):
Reference Control
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 10/157-023DA) with reference item Potassium dichromate (batch no.: 0769128) was: 23 - 24 June 2010.
The 24h EC50: 1.77 mg/L, (95 % confidence limits: 1.46 – 2.15 mg/L)
This value is within the range of laboratory ring test data (see ISO Guideline No. 6341).
Reported statistics and error estimates:
No statistical analysis was necessary because the lack of toxic effects. The EC50, NOEC and LOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Acute toxicity of the test material was assessed with acute immobilisation test on daphnia (Daphnia magna), over an exposure period of 48 hours in a semi-static system.
Under the conditions of this Daphnia magna acute immobilisation study, 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 item had no toxic effect at saturation.
The observed endpoints for the effect of the test material were the following:
The 24h EC50 value: >0.3 mg/L
The 48h EC50 value: >0.3 mg/L
The 48h NOEC value: 0.3 mg/L
The 48h LOEC value: >0.3 mg/L
The results above were based on the measured concentration and determined directly from the raw data.
Executive summary:

Acute toxicity of the test material was assessed with acute immobilisation test on Daphnia magna (10/095-023DA), over an exposure period of 48 hours in a semi-static system according to EU Test Method C.2.

Under the conditions of this Daphnia magna acute immobilisation study, 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 item had no toxic effect at saturation.

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

The 24h EC50 value: >0.3 mg/L

The 48h EC50 value: >0.3 mg/L

The 48h NOEC value: 0.3 mg/L

The 48h LOEC value: >0.3 mg/L

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