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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Analytical monitoring:
yes
Details on sampling:
For the analysis of the actual test item concentrations, the following samples were taken:
- just before the start: duplicate samples from the single test medium (undiluted filtrate of a supersaturated dispersion with a loading rate of 100 mg/L, without daphnids) and duplicate samples from the control (without daphnids)
- after 48 hours (stability samples): duplicate samples from the single test medium (without daphnids) and duplicate samples from the control (without daphnids)

For the 48-hour stability samples, the contents of the two respectiv test beakers were combined.
All the samples were analyzed immediately without prior storage.

The concentration of the test substance was analyzed in the duplicate test medium samples of the single test concentration of nominal 100 mg/L for both sampling times (0 and 48 hours). From the control only one of the duplicate samples was analzed from each of both sampling times.
Vehicle:
no
Details on test solutions:
According to the results of a pre-experiment (without GLP), the test item was not soluble at a concentration of 100 mg/L in test water and no homogeneous dispersion could be prepared.

An undiluted filtrate of a supersaturated dispersion with the maximum concentration of dissolved test item was used as the only test concentration. Additionally, a control was tested in parallel (test water without test item).
The test medium was prepared as follows:
a supersaturated dispersion with a loading rate of 100 mg/L was prepared by weighing 33 mg of the test item into 300 mL test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible by ultrasonic treatment for 15 minutes and by intense stirring for 3 hours at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion. The stirring period of 3 hours was chosen according to the results of a pre-test (without GLP) which showed that the solution equilibrium was reached after this time. In this pre-test the same test item concentrations were analytically measured in filtrates after stirring for 3, 24 and 96 hours.
Then, the supersaturated dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm) just before the preparation of the test medium.
The actual concentrations of the test item in the test medium were analytically determined. The test medium was prepared just before introduction of daphnids (= start of the test).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- The study was performed with young daphnids of a clone of the species Daphnia magna Straus. A clone of this species was originally supplied by the University of Sheffield/UK in 1992, defined from the supplier as clone 5. Since the date the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (regarding pH, main ions and total hardness) and under temperature and light conditions identical to those of the tests.
- Age at study initiation (mean and range, SD): at the start of the test the used test organisms were 6-24 hours old and were not first brood progeny.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
2.5 mmol/L (= 250.0 mg/L) as CaCO3
Test temperature:
21°C
pH:
7.9
Dissolved oxygen:
8.0-8.3 at the start of the test (t = 0h) and 8.1 at the end of the test (t = 48h)
Nominal and measured concentrations:
Nominal concentration: undiluted filtrate of a supersaturated dispersion with a loading rate of 100 mg/L
Measured concentration: below the limit of quantification (= 0.0643 mg/L)
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 mL glass beakers filled with 50 mL test medium. The beakers were covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Loading rate: < 1 daphnia/2 mL test solution
- Feeding: none

TEST MEDIUM / WATER PARAMETERS
- Reconstituted test water: analytical grade salts were dissolved in purified water to obtain the following nominal concentrations:
- CaCl2 x 2H2O: 2.0 mmol/L (= 294 mg/L)
- MgSO4 x 7 H2O: 0.5 mmol/L (= 123 mg/L)
- NaHCO3: 0.75 mmol/L (= 65 mg/L)
- KCl: 0.075 mmol/L (= 5.8 mg/L)
- Alkalinity: 0.8 mmol/L
- Ratio of Ca:Mg: 4:1 (based on molarity)
- Ratio of Na:K: 10:1 (based on molarity)
The test water was aerated until oxygen saturation was obtained. During the test period, the test water was not aerated.

OTHER TEST CONDITIONS
- Photoperiod: The test was performed as far as possible in the dark to avoid photolytic degradation of the test item.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- The daphnids were observed for immobility after 24 and 48 hours of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.

TEST CONCENTRATIONS
- According to the results of a pre-experiment (without GLP), the test item was not soluble at a concentration of 100 mg/L in test water and no homogeneous dispersion could be prepared.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: No acute toxic effects within the range of solubility.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: No acute toxic effects within the range of solubility.
Details on results:
The analytically measured test item concentrafions in the analyzed test medium samples (the undiluted filtrate of the supersaturated dispersion) were below the limit of quantification of the analytical method (LOQ, 0.0643 mg/L). Therefore, the biological results are related to the loading rate of 100 mg/L.
In the control no immobilized test organisms or other signs of intoxication were determined during the test period of 48 hours. In the test medium two test organisms were immobile at the observation after 24 hours. However, this immobilization rate was not estimated as a significant toxic effect, because according to the test guidelines an immobilization rate of 10% is tolerated in the control. Furthermore, no immobilization was observed in a pre-test (without GLP).
Therefore, the 48-hour NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours), respectively the 48-hour ECO of the test substance to Daphnia magna were determined to be at least at a loading rate of 100 mg/L. The 48-hour NOEC and the 48-hour ECO might even be higher, but loading rates in excess of 100 mg/L have not been tested according to the EU Commission Directive. The 48-hour EC50 and the 48-hour EC100 were clearly above the loading rate of 100 mg/L. These values could not be quantified, since the test item had no toxic effect at the loading rate of 100 mg/L, and thus above the water solubility limit of the test item in the used test water.

In conclusion, the test item had no acute toxic effects on Daphnia magna up to the water solubility limit.

At the beginning and the end of the test period, the dissolved oxygen concentrations in the test media and the control were at least 8.0 mg/L. The pH value was 7.9 and water temperature was constantly 21 °C.
No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the whole test duration.
Validity criteria fulfilled:
yes
Conclusions:
No acute toxic effects occur within the range of solubility. The test substance is with high probability not acutely harmful to aquatic invertebrates.
Executive summary:

In this guideline (OECD 202) study, the 48 hour EC50 of the test material (EC 438-340-0) to Daphnia magna was determined to have no acute toxic effects within the range of solubility. The test was conducted under static conditions. The result of this test is not sufficient to trigger classification and labelling under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Description of key information

Study conducted to recognised testing guidelines with GLP.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

No acute toxic effects occur within the range of solubility. The test substance is with high probability not acutely harmful to aquatic invertebrates. The study was performed according to OECD guideline as a limit test with 100 mg/L (RCC Ltd. 2002i). Due to the low solubility of the test substance in the test medium a saturated solution has been used. The actual measured concentration was below the limit of quantification of the analytical method (0.0643 mg/L) at the start and at the end of the test medium renewal periods. The EC50and EC100are both higher than 100 mg/L (loading rate). No mortality or visible abnormalities were observed during the test period of 48 hours (NOEC is at least 100 mg/L).

The study was performed according to OECD guideline as a limit test with 100 mg/L (RCC Ltd. 2002i). Due to the low solubility of the test substance in the test medium a saturated solution has been used. The actual measured concentration was below the limit of quantification of the analytical method (0.0643 mg/L) at the start and at the end of the test medium renewal periods. The EC50and EC100are both higher than 100 mg/L (loading rate). No mortality or visible abnormalities were observed during the test period of 48 hours (NOEC is at least 100 mg/L).

The study was performed according to OECD guideline as a limit test with 100 mg/L (RCC Ltd. 2002i). Due to the low solubility of the test substance in the test medium a saturated solution has been used. The actual measured concentration was below the limit of quantification of the analytical method (0.0643 mg/L) at the start and at the end of the test medium renewal periods. The EC50and EC100are both higher than 100 mg/L (loading rate). No mortality or visible abnormalities were observed during the test period of 48 hours (NOEC is at least 100 mg/L).