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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:
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:
Stored frozen at approximately -20 ºC in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each 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.
Only samples at the No Observed Effect Concentration were analyzed.
Vehicle:
yes
Details on test solutions:
Reconstituted Water – ISO Medium

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:
None
pH:
7.8 ± 0.2
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 the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.
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 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 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.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L. A nominal amount of test item (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 56, 32, 18 and 10 mg/L. Each 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
In the definitive test 150 mL glass jars containing approximately 100 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 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. The test preparations were not renewed during the exposure period.
Reference substance (positive control):
yes
Remarks:
A positive control (Envigo Study Number 41502271) used potassium dichromate as the reference item. The positive control was conducted between 21 September 2015 and 23 September 2015.
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:
After 48 hours exposure, no immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, 50% immobilization was observed at 100 mg/L. Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test. Chemical analysis of the 10 and 100 mg/L test preparations at 0 and 48 hours showed near nominal concentrations were obtained indicating that the test item was stable over the test duration.

Definitive Test:
Verification of Test Concentrations
Analysis of the 100 mg/L 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.

Immobilization Data
There was no significant immobilization in 20 daphnids exposed to a maximum 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
A single daphnid was observed to be trapped at the surface in the 18 mg/L test concentration after 24 hours exposure. After 48 hours exposure daphnids showing reduced mobility were observed in all 100 mg/L test preparations.

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 20 °C to 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 424 to 510 Lux.

Observations on Test Item Solubility
At the start and throughout the test all control solutions were observed to be clear colorless solutions. The test cultures were observed to range from pink solutions at 10 mg/L through to dark red solutions at 100 mg/L throughout the duration of the test.
Results with reference substance (positive control):
None
Reported statistics and error estimates:
None

None

Validity criteria fulfilled:
yes
Conclusions:
The 48 hour EC50 and NOEC values were determined to >100 mg/L and 100 mg/L, respectively.
Executive summary:

A key study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guideline 202 and EU Method C.2. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 20 °C to 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours. Analysis of the 100 mg/L 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. There was no significant immobilization in 20 daphnids exposed to a maximum test concentration of 100 mg/L for a period of 48 hours. Hence, the 48 hour EC50 and NOEC values were determined to >100 mg/L and 100 mg/L, respectively.

Description of key information

The 48 hour EC50 and NOEC values were determined to >100 mg/L and 100 mg/L, respectively.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

A key study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guideline 202 and EU Method C.2. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 20 °C to 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours. Analysis of the 100 mg/L 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. There was no significant immobilization in 20 daphnids exposed to a maximum test concentration of 100 mg/L for a period of 48 hours. Hence, the 48 hour EC50 and NOEC values were determined to >100 mg/L and 100 mg/L, respectively.