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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:
22 February 2016 to 14 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP 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)
Specific details on test material used for the study:
- Physical state: Yellow semi-solid
- Analytical purity: 100 %
- Expiration date of the lot/batch: 23 November 2017
- Storage condition of test material: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
RANGE-FINDING TEST
- 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.

DEFINITIVE TEST
- Samples were taken from the control and each test group at 0 hours from the bulk test preparation and at 48 hours from the pooled replicates for quantitative analysis.
- 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:
no
Details on test solutions:
TEST WATER
- Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined in Annex 4 (attached).

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 mg/L.
- 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 test concentrations of 0.10, 1 and 10 mg/L.
-Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.


DEFINITIVE TEST
- Based on the result of the range-finding test the following test concentrations were assigned to the definitive test: 6.2, 12.5, 25, 50 and 100 mg/L.
- A nominal amount of test item (200 mg) was dissolved in test water with the aid of ultrasonication for approximately 1 hour and the volume adjusted to 2 L to give the 100 mg/L test concentration from which a series of dilutions was made to give test concentrations of 6.2, 12.5, 25 and 50 mg/L.
- Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
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 (see Annex 2, attached) in a temperature controlled room maintaining water temperature at 18 to 22°C.
- The lighting cycle was controlled to give 16 hours light and 8 hours darkness with 20 minute dawn and dusk 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 so that 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 were 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:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
250 mg/L total theoretical hardness as CaCO3
Test temperature:
21 °C
pH:
7.6 to 7.9
Dissolved oxygen:
8.6 to 9.4 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
RANGE-FINDING TEST
- Nominal 0.10, 1.0, 10 and 100 mg/L

DEFINITIVE TEST
- Nominal 6.2, 12.5, 25, 50 and 100 mg/L
Details on test conditions:
RANGE-FINDING TEST
- Daphnids (10) were placed in each test and control vessel and maintained in a temperature controlled room maintaining 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 immobilised daphnids were recorded.
- The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
- 150 mL glass beakers containing approximately 100 mL of test preparation were used.
- At the start of the test daphnids (5) were placed in each test and control vessel at random.
- Four replicate test and control vessels were prepared.
- Test vessels were covered to reduce evaporation and maintained in a temperature controlled room maintaining water temperature at 18 to 22 °C with a maximum deviation of ± 1°C with a photoperiod of 16 hours light (200 to 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.
- 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:
potassium dichromate conducted between 07 September 2015 and 23 September 2015 (see Annex 3, attached)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (e.g. 95% CL)
Remarks:
Not possible to determine
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (e.g. 95% CL)
Remarks:
17 - 25 mg/mL
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST
- Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (attached).
- No immobilisation was observed at 0.10 and 1.0 mg/L, however significant immobilisation observed at 10 and 100 mg/L.
- Based on this information, test concentrations of 6.2, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.
- Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 92 to 149% of nominal concentrations. (See Annex 5, attached).
-At 48 hours, no significant change in the measured concentrations obtained for the 10 and 100 mg/L test preparations indicating that the test item was stable under test conditions at these concentrations.
-Significant declines in the measured concentrations at 48 hours for the 0.10 and 1.0 mg/L test preparations were observed
-Given that the toxic range of the test item was in excess of these concentrations this did not affect the conduct of the definitive test.

DEFINITIVE TEST – VERIFICATION OF TEST CONCENTRATIONS
- Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal. As such the results are based on nominal test concentrations (see Annex 5, attached).

DEFINITIVE TEST IMMOBILISATION DATA
- Cumulative immobilisation data and other observations from exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached).
- The relationship between percentage immobilization and concentration at 48 hours is given in Figure 1 (attached).
- Analysis of immobilization data by Probit analysis using Linear Maximum-Likelihood regression based on nominal test concentrations gave the following results:
-The EC50 after 24 and 48 hours were 55 and 20 mg/L (with 95% confidence limits of "Not possible to determine for 24h and 17-25 mg/L for 48h), respectively.
-The NOEC after 24 and 48 hours exposure were 50 and 12.5 mg/L respectively.
-The LOEC after 24 and 48 hours exposure were 100 and 25 mg/L respectively.
- The slopes and their standard errors of the response curves at 24 and 48 hours were 5.5 (SE = 0.04) and 4.4 (SE = 0.04) respectively.

DEFINITIVE TEST – Sub-Lethal Effects
-Sub-lethal effects of exposure were observed in the 12.5, 25 and 50 mg/L test concentrations.
-The sub-lethal response was reduced mobility (see Table 2, attached).

DEFINITIVE 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 ≥ 3 mg/L in the control and test vessels.

DEFINITIVE TEST - WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 21 to 22 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.
- Light intensity was observed to be in the range 507 to 620 lux throughout the test.

DEFINITIVE TEST - OBSERVATIONS ON TEST ITEM SOLUBILITY
- At the start and throughout the test all control and test solutions were observed to be a clear colourless water solutions.

Validity criteria fulfilled:
yes
Conclusions:
Exposure of freshwater invertebrate Daphnia magna has been investigated and gave the following results:
EC50 (48h) = 20 mg/L (with 95% confidence limits of 17-25 mg/L); NOEC (48h) = 12.5 mg/L; LOEC (48h) = 25 mg/L.
Executive summary:

INTRODUCTION

A 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 Guidelines for Testing of Chemicals (April 2004) No 202 "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No 440/2008.

METHODS

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 6.2, 12.5, 25, 50 and 100 mg/L for 48 hours at a temperature of 21 to 22°C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

RESULTS

Analysis of the test preparations at 0 and 48 hours shoed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item the following results:

EC50 (48h) = 20 mg/L (with 95% confidence limits of 17-25 mg/L); NOEC (48h) = 12.5 mg/L; LOEC (48h) = 25 mg/L.

Description of key information

Exposure of freshwater invertebrate Daphnia magna has been investigated and gave the following results: EC50 (48h) = 20 mg/L (95% confidence limits of 17 – 25 mg/L) based on analytically confirmed nominal concentrations. (OECD 202 and EU Method C.2.)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
20 mg/L

Additional information

INTRODUCTION

A 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 Guidelines for Testing of Chemicals (April 2004) No 202 "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No 440/2008.

METHODS

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 6.2, 12.5, 25, 50 and 100 mg/L for 48 hours at a temperature of 21 to 22°C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

RESULTS

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.

Exposure of Daphnia magna to the test item the following results:

EC50 (48h) = 20 mg/L (95% confidence limits of 17 – 25 mg/L); NOEC = 12.5 mg/L and LOEC = 25 mg/L