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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:
27 February 2017 - 01 August 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Verification of Test Concentrations
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:
not specified
Details on test solutions:
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions

A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of deionized 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 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)


Examination of the centrifuged test samples showed that tiny particles of undissolved test item remained, as such the use of centrifugation was considered inappropriate for this test item. There was no significant increase the dissolved test item concentration obtained from the filtered samples when extending the stirring period beyond 24 hours.
Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 0.23 mg/L. A reduction in the initial loading rate to 50 mg/L would still ensure a sufficient excess of test item is present to allow complete saturation of the test media.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 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. 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.
Test type:
static
Water media type:
not specified
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
21°C to 23°C
pH:
7.9 ± 0.3
Dissolved oxygen:
>3 mg/L
Nominal and measured 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.
Definitive test- Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at highest attainable test concentration no immobilization or adverse reactions to exposure were observed.
Details on test conditions:
Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.23 mg/L could be obtained using a saturated solution method of preparation.
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
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.
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel 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. 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 item.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at highest attainable test concentration no immobilization or adverse reactions to exposure were observed.
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 (old and fresh media) and 48 hours

Exposure Conditions
As in the range-finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 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.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. 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.

Test Organism Observations
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.





Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Details on results:
Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

At 24 hours the 0.10 and 10% v/v saturated solutions were observed to have a significant amount of debris in the water column.
At 48 hours all test solutions, including the control, were observed to have a significant amount of debris in the water column. This was not considered to have affected the validity or integrity of the study as there were no effects on the daphnia in any test concentration.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 immobilization or adverse reactions to exposure were observed. Chemical analysis of the 100% v/v saturated solution test preparation at 0 hours showed measured test concentration of 0.15 mg/L. There was a significant decline in the measured concentration at 48 hours indicating that the test item was not stable under test conditions.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to be 0.25 and 0.22 mg/L, respectively. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined to 0.13 and 0.12 mg/L (52% to 54% of the fresh measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.
The geometric mean measured test concentration was determined to be 0.18 mg/L


Immobilization Data
There was no significant immobilization in 20 daphnids exposed to a test concentration of 0.18 mg/L for a period of 48 hours.
Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.18 mg/L. The No Observed Effect Concentration was 0.18 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 immobilization 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
The results of the water quality measurements are given in Table 3. Temperature was maintained at 21°C to 23 °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 530 to 692 Lux.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):
A positive control (Envigo Study Number XT01HS) 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 immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

24 hours-EC50-1.3mg/L
48 hours-EC50-1.2mg/L

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization 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:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the geometric mean measured test concentrations gave a 48-Hour EC50 value of greater than 0.18 mg/L. The No Observed Effect Concentration was 0.18 mg/L.
This study showed that there were no toxic effects at saturation.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.23 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item 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 item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of 21°C to 23°C under static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.

Results

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to be 0.25 and 0.22 mg/L, respectively. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined to 0.13 and 0.12 mg/L (52% to 54% of the fresh measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.18 mg/L.

Exposure ofDaphnia magnato the test item gave EC50values based on the geometric mean measured test concentrations of greater than 0.18 mg/L. The No Observed Effect Concentration was 0.18 mg/L.

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

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the geometric mean measured test concentrations gave a 48-Hour EC50value of greater than 0.18 mg/L. The No Observed Effect Concentration was 0.18 mg/L.

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

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.18 mg/L
EC50/LC50 for marine water invertebrates:
0.18 mg/L

Additional information