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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:
14 June 2017 to 27 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Samples were taken from the control and the 100 % v/v saturated solution test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis.
- All samples were stored frozen prior to analysis.
- Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Remarks:
Test material dispersed in deionised reverse osmosis water
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test material was categorised as being a ‘difficult substance’ as defined by OECD. Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
- In the trial a nominal amount of test material (1100 mg) was dispersed, in duplicate, in 11 litres of deionised reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments: centrifugation at 10000 g for 30 minutes, centrifugation at 40000 g for 30 minutes, filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter) and filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 litre discarded in order to pre-condition the filter).
- Based on the preparation trial the test material was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 litre discarded) to give a nominal test concentration of approximately 0.34 mg/L
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In house
- Age: 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.
- Feeding during test: no

CULTURE CONDITIONS
- Acclimation conditions: Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °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.
- Feeding: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and flake food suspension.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
22 °C
pH:
7.6 to 7.9
Dissolved oxygen:
8.0 to 8.6 mg O2/L
Nominal and measured concentrations:
Nominal: 100 % v/v saturated solution
Measured: 0.43 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass beakers
- The test vessels were covered to reduce evaporation
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO media) was used for both the range-finding and definitive tests and contained: CaCl2.2H2O 294 mg/L, MgSO4.7H2O 123 mg/L, NaHCO3 65 mg/L and KCl 5.8 mg/L.
- Culture medium different from test medium: yes culture medium was Elendt M7 medium
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Light intensity: between 200 and 1200 Lux
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED:
- Test Organism Observations: Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution.
- In the range-finding test, two replicate vessels each containing 5 daphnids were used per test group and maintained in a temperature controlled room maintaining the water temperature at 18 to 22°C with a maximum deviation of ±1°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded. The control group was maintained under identical conditions but not exposed to the test material.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST
- No significant immobilisation was observed at the test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution.
- A sub-lethal effect of exposure was observed in the 1.0 % v/v saturated solution test concentration. This response was trapping at the surface.
- Based on this information, a single test concentration of four replicates, of 100 % v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.
- Chemical analysis of the 100 % v/v saturated solution test preparation at 0 hours showed a measured test concentration of 0.29 mg/L. There was a significant decline in the measured concentrations at 48 hours indicating that the test material was not stable under test conditions.

DEFINITIVE TEST
- Verification of Test Concentrations: Analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to be 0.44 to 0.61 mg/L respectively. A decline in measured test concentration was observed in the old or expired media at 24 and 48 hours to 0.29 and 0.43 mg/L respectively and hence it was considered appropriate to calculate the results based on the mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.43 mg/L.
- Immobilisation Data: There was no immobilisation in 20 daphnids exposed to a test concentration of 0.43 mg/L for a period of 48 hours. Exposure of Daphnia magna to the test material gave EC50 values based on the geometric mean measured test concentration of greater than 0.43 mg/L. The No Observed Effect Concentration was 0.43 mg/L.
- Sub-Lethal Effects: No sub-lethal effects of exposure were observed throughout the test.
- Validation Criteria: The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
- Water Quality Criteria: Temperature was maintained at 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 531 to 561 Lux.
- Observations on Test Material Solubility: At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.
Results with reference substance (positive control):
- A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Exposure conditions for the positive control were similar to those in the definitive test.
- Analysis of the immobilisation data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results: 48-h EC50: 0.64 mg/L, 48-h NOEC: 0.56 mg/L.
- The No Observed Effect Concentration is based upon equal to or less than 10 % immobilisation at this concentration. The results from the positive control with potassium dichromate were within the normal range for this reference item.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the exposure of Daphnia magna to the test material gave EC50 values based on the geometric mean measured test concentration of greater than 0.43 mg/L. The No Observed Effect Concentration was 0.43 mg/L. This study showed there were no toxic effects as saturation.
Executive summary:

The acute toxicity of the test material to aquatic invertebrates was determined in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions using Daphnia magna.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.34 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a nominal test concentration of 100 % v/v saturated solution for 48 hours at a temperature of 22 °C under semi-static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material.

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations of 0.44 and 0.61 mg/L respectively. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to 0.29 and 0.43 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured concentration was calculated to be 0.43 mg/L.

This study showed that there were no toxic effects at saturation.

Under the conditions of this study, the exposure of Daphnia magna to the test material gave EC50 values based on the geometric mean measured test concentration of greater than 0.43 mg/L. The No Observed Effect Concentration was 0.43 mg/L. This study showed there were no toxic effects as saturation.

Description of key information

Under the conditions of the study, the exposure of Daphnia magna to the test material gave EC50 values based on the geometric mean measured test concentration of greater than 0.43 mg/L. The No Observed Effect Concentration was 0.43 mg/L. The study showed there were no toxic effects as saturation.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.43 mg/L

Additional information

The acute toxicity of the test material to aquatic invertebrates was determined in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions using Daphnia magna. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.34 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a nominal test concentration of 100 % v/v saturated solution for 48 hours at a temperature of 22 °C under semi-static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material.

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations of 0.44 and 0.61 mg/L respectively. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to 0.29 and 0.43 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured concentration was calculated to be 0.43 mg/L.

This study showed that there were no toxic effects at saturation.

Under the conditions of this study, the exposure of Daphnia magna to the test material gave EC50 values based on the geometric mean measured test concentration of greater than 0.43 mg/L. The No Observed Effect Concentration was 0.43 mg/L. This study showed there were no toxic effects as saturation.