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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:
From 17-Oct-2008 to 12-Dec-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 202 and EU Method C.2 with GLP statement. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. All validity criteria were fulfilled.
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2008-11-12
Specific details on test material used for the study:
- Storage condition of test material: in a refrigerator at about 4°C.
- Water solubility: 1.88 mg/L
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The test item (53.02 mg) was dissolved in ethyl acetate and made up to the mark in a 50 mL volumetric flask to prepare a stock solution with a concentration of 1060 mg/L. Thereof, 1 mL were diluted to 10 mL using ethyl acetate to obtain a working solution with a concentration of
106 mg/L.
- Sample storage conditions before analysis: the samples were stored deep-frozen and protected from light until analysis was performed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 100.6 mg of the test item in 1000 mL of test water. Ultrasonic treatment for 15 minutes and intense stirring were applied. No auxiliary solvent or emulsifier was used. The dispersion was stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum amount of the test item in the dispersion. After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The undiluted filtrate of the dispersion with the loading rate of 100 mg/L was used as test medium. The test medium was prepared just before the start of the test (=addition of daphnids).
- Eluate: test water
- Controls: one control (test water without addition of the test item)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnids
- Strain: no data
- Source: a clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992.
- Age at study initiation (mean and range, SD): 6-24 hours old
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Valve height at study initiation, for shell deposition study (mean and range, SD): no data
- Peripheral shell growth removed prior to test initiation: no data
- Method of breeding: the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Feeding during test: no
- Food type: not applicable
- Amount: not applicable
- Frequency: not applicable

ACCLIMATION
no data

QUARANTINE (wild caught)
not applicable
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
The water hardness was 240 mg/L as CaCO3.
Test temperature:
The water temperature during the test was 20°C.
pH:
The pH values of the test medium and control were between 7.8 to 7.9.
Dissolved oxygen:
At the beginning and end of the test period, the dissolved oxygen concentration in the test medium and control was at least 8.1 mg/L (8.1-8.6 mg/L).
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentration: loading rate of 100 mg/L
Measured concentrations: 2.2 mg/L (start of the test); 1.6 mg/L (end of the test). The mean measured concentration was 1.8 mg/L (calculated as geometric mean).
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: 100 mL glass beakers.
- Aeration: During the test period, the test water was not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: the volume of test solution provided for each daphnia was 10 mL.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted test water according to ISO 6341 was used in this study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations: CaCl2 x 2H2O = 294 mg/L ; MgSO4 x 7H2O = 123 mg/L; NaHCO3 = 65 mg/L; KCl = 5.8 mg/L; Water Hardness = 250 mg/L as CaCO3; Alkalinity = 0.8 mmol/L. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the preparation of the test medium until oxygen saturation was reached. During the test period, the test water was not aerated.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: a 16-h light to 8-h dark photoperiod with a 30-min transition period was used
- Light intensity: within the range of 500 to 630 Lux

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes (non-GLP)
- Results used to determine the conditions for the definitive study: no data
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: The highest concentration without observed effect
Effect conc.:
>= 1.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
At the start of the test, the measured concentration of the test item in the analyzed test medium amounted to 2.2 mg/L. At the end of the test, the
measured concentration was 1.6 mg/L resulting in a mean measured concentration of 1.8 mg/L (calculated as geometric mean).

In the control and at the loading rate of 100 mg/L (measured concentration of 1.8 mg/L) no immobilized daphnids were determined during the test period of 48 hours. See table 6.1.3/1 in "Any other information on results incl. tables".

No remarkable observations were made concerning the appearance of the test medium. The test medium was clear throughout the whole test duration.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50/LC50: The result of the latest positive control test in March 2008 (48-hour EC50: 0.73 mg/L, study B83744) indicated that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 1996 to 2008: 0.53-1.1 mg/L).
- Other: none
Reported statistics and error estimates:
None

Table 6.1.3/1: Effect of the test item on the mobility of Daphnia magna

Loading rate (mg/L)

Mean measured test item concentration (mg/L)

No. of daphnids tested

Immobilized daphnids after 24 hours

Immobilized daphnids after 48 hours

No.

%

No.

%

Control

100

Control

1.8

20

20

0

0

0

0

0

0

0

0

Validity criteria fulfilled:
yes
Conclusions:
The test item had no toxic effects on Daphnia magna up to its water solubility limit in test water at a loading rate of 100 mg/L (measured concentration of 1.8 mg/L). The highest concentration without observed effect after 48 hours was >= 1.8 mg/L and the 48-hour EC50 was > 1.8 mg/L.
Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

 

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the daphnids up to the loading rate of 100 mg/L.

 

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered through a 0.45 μm membrane filter. The undiluted filtrate of the dispersion was used as the test medium. Additionally, a control was tested in parallel. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

At the start of the test, the measured concentration of the test item in the analyzed test medium amounted to 2.2 mg/L. At the end of the test, the measured concentration was 1.6 mg/L resulting in a mean measured concentration of 1.8 mg/L (calculated as geometric mean). The biological results were based on the loading rate of 100 mg/L and on the mean measured test item concentration of 1.8 mg/L.

 

In the control and at the loading rate of 100 mg/L (measured concentration of 1.8 mg/L) no immobilized daphnids were determined during the test period of 48 hours.

 

Therefore, based on measured concentrations, the highest concentration without observed effect after 48 hours was >= 1.8 mg/L and the 48-hour EC50 was > 1.8 mg/L. These values could not be quantified due to the absence of toxicity of the test material at the tested loading rate.

 

In conclusion, the test item had no toxic effects on Daphnia magna up to its water solubility limit in test water at a loading rate of 100 mg/L.

Description of key information

EU Method C.2, OECD Guideline 202, GLP, key study, validity 1:

No acute toxic effects up to its water solubility limit in test water at a loading rate of 100 mg/L, corresponding to a geometric mean measured concentration of 1.8 mg/L. The 48-hour EC50 (Daphnia magna) was > 1.8 mg/L.

Key value for chemical safety assessment

Additional information

One valid key study is available to assess the toxicity of the substance to aquatic invertebrates Daphnia magna, according to Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004) with GLP statement.

In this study, a limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the daphnids up to the loading rate of 100 mg/L. Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered through a 0.45 μm membrane filter. The undiluted filtrate of the dispersion was used as the test medium. Additionally, a control was tested in parallel. At the start of the test, the measured concentration of the test item in the analyzed test medium amounted to 2.2 mg/L. At the end of the test, the measured concentration was 1.6 mg/L resulting in a mean measured concentration of 1.8 mg/L (calculated as geometric mean). In the control and at the loading rate of 100 mg/L no immobilized daphnids were determined during the test period of 48 hours.

Therefore, based on measured concentrations, the highest concentration without observed effect after 48 hours was >= 1.8 mg/L and the 48-hour EC50 was > 1.8 mg/L. These values could not be quantified due to the absence of toxicity of the test material at the tested loading rate.

In conclusion, the test item had no toxic effects on Daphnia magna up to its water solubility limit in test water at a loading rate of 100 mg/L.