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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:
05 November 2013 to 15 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Based on OECD test guideline and GLP compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: off-white powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
A sample of each test concentration was taken for chemical analysis at O and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Test solutions

Vehicle:
not specified
Details on test solutions:
Reagents and Solvents
Water: prepared using an ELGA Pure Lab Option R-15 water purification system.

Solvents: acetonitrile, Fisher Scientific, HPLC gradient grade.

Reagents: orthophosphoric acid, Fisher Scientific, Laboratory reagent grade

Preparation of Calibration Standards
The test item (nominal 100 mg) was dissolved in acetonitrile (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. This stock solution was further diluted with acetonitrile:water (50:50 v/v) to obtain a nominal 10 mg/L calibration standard. A duplicate calibration standard was similarly prepared at 10 mg/L. These duplicate calibration standards were used to determine the recovery and test sample concentrations.

Preparation of Linearity Standards
The test item (nominal 100 mg) was dissolved in acetonitrile (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. Defined volumes of this stock solution were diluted with acetonitrile:water (50:50 v/v) to obtain standards in the range of 0.05 to 30 mg/L. A second standard was similarly prepared at a nominal concentration of 10 mg/L. These standards were used to evaluate the linearity of the analytical system.

Preparation of Spiked Recovery Samples
To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in acetonitrile to prepare a stock solution with a concentration of 1000 mg/L. This
stock solution was further diluted with acetonitrile to produce a stock solution of 500 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 1 mg/L. Five replicates at this concentration level were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

Preparation of Test Samples
The 0-Hour test samples were thawed with the aid of sonication, whilst the 48-Hour test samples were analyzed on the day of receipt. A defined volume of test sample (50 mL) was diluted with 50 mL of water in a 200 mL volumetric, and then made to volume with acetonitrile. An aliquot was then taken for analysis. See Table 1 for more information on preparation volumes.

Test organisms

Test organisms (species):
Daphnia sp.
Details on test organisms:
Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems.

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

A positive control (Harlan Study Number 41301833) used potassium dichromate as the reference item.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/Las CaC03.
Test temperature:
20 °C
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value. The dissolved oxygen concentration at the end of the test is 2:3 mg/Lin the control and test vessels.
Nominal and measured concentrations:
Nominal concentrations: 1.0, 3 .2, 10, 32 and 100 mg/L.

Chemical analysis of the test preparations at O and 48 hours showed measuredconcentrations of between 71 % and 93 % of nominal.
Details on test conditions:
As in the range-finding test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared.
The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light (595 to 798 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period.

Due to the fact that a flat response was observed and no EC50 value was obtained in the initial tests, the definitive test was conducted using the following range of nominal concentrations: 1.0, 3 .2, 10, 32 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 7.8 - 13 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 7.8 - 13 mg/L
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 7.8 - 13 mg/L
Details on results:
Analysis of the 3.2, 10, 32 and 100 mg/L test preparations at O and 48 hours showed measured test concentrations to range from 88% to 93% of nominal value and so the results are based on nominal test concentrations only.
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
Statistical Analysis
The EC50 values and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999) and at 24 hours using the geometric mean method as follows:
ECso value= the square root of C1 x C2

Where:
C1 = concentration showing 0% immobilization
C2 = concentration showing 100% immobilization
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.
If there is no immobilization between 0% and 100% immobilization, then the geometric mean of the highest test concentration showing no lethality and the lowest test concentration showing 100% lethality is calculated. The concentrations resulting in 0% and 100% immobilization will
be the 95% confidence limits.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item has been investigated and gave the following results:

Time Point EC 50 95% Confidence Limits No Observed Effect Concentration Lowest Observed Effect
(Hours) (mg/L) (mg/L) (NOEC) (mg/L) Concentration (LOEC) mg/L

48 10 7.8 - 13 3.2 10