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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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
Storage Conditions: Approximately -20 ºC in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
Vehicle:
yes
Details on test solutions:
Reconstituted water (ISO medium) used for both the range-finding and definitive tests.

Ingredient Final Concentration (mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 65
KCl 5.8

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
Test organisms (species):
Daphnia magna
Details on test organisms:
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 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:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
250 mg/L as CaCO3
Test temperature:
22 °C
pH:
Control: 7.8
Test: 7.9
Dissolved oxygen:
Approximately air-saturation value
Salinity:
None
Conductivity:
None
Nominal and measured concentrations:
Range-finding Test: 0.10, 1.0, 10 and 100 mg/L.
Definitive Test: Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.
Details on test conditions:
Range-finding Test:
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 1.0 and 0.10 mg/L.
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 photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 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 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.
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 were verified by chemical analysis at 0 and 48 hours.

Exposure 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 maintaining the water temperature at 18 to 22 °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.
The test preparations were not renewed during the exposure period.

Assessments
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.
Water Quality Criteria
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. 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 light intensity during the light period was measured using an ATP Instruments Lux meter.
Verification of Test Concentrations
Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in Annex 4.
Data Evaluation
Statistical Analysis
An estimate of the EC50 values was given by inspection of the immobilization data.
Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
• No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
• The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.
Reference substance (positive control):
yes
Remarks:
A positive control (Envigo Study Number 41502271) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Based on this information, a single test concentration of four replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.
Chemical analysis of the 100 mg/L test preparations at 0 and 48 hours showed measured test concentrations to be near nominal indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 98 % to 100 % of nominal concentrations and so the results are based on nominal test concentrations.
Immobilization Data
There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EC50 (mg/L) 95 % Confidence limits (mg/L)
24 >100 NA
48 >100 NA

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 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
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 534 to 694 Lux.

Observations on Test Item Solubility
At the start and throughout the test all control solutions were observed to be clear colorless solutions whilst the 100 mg/L test solutions were observed to be very dark red opaque solutions.
Results with reference substance (positive control):
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization 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:

Time Point (Hours) EC50 (mg/L) 95 % Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
24 0.90 Not possible to determine 0.56 1.0
48 0.54 0.47-0.63 0.32 0.56

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.
Reported statistics and error estimates:
None

None

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to FAT 40032/G gave EC50 value of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.
Executive summary:

A GLP study was performed to assess the acute toxicity of the test item to Daphnia magna according to the OECD Guideline 202 and EU Method C.2.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals each) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of approximately 21 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.  

Exposure of Daphnia magna to the test item gave EC50value of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Description of key information

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

The key GLP study was performed to assess the acute toxicity of the test item to Daphnia magna according to the OECD Guideline 202 and EU Method C.2.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals each) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of approximately 21 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.  

Exposure of Daphnia magna to the test item gave EC50 value of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.