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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:
2017-09-04 to 2017-11-21
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:
April 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EC No 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 2018-12-30
- Purity: 98.2%
- Purity test date: 2017-01-04 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
Analytical monitoring:
yes
Details on sampling:
- Sampling method: 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. Only the concentration to be used for the initial experiment was analyzed.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
* Range-finding Test: The test concentration to be used in the definitive test was determined by a preliminary 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 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
* 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 10, 18, 32 and 56 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water-flea
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): 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 cultivation: 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.
- Feeding during the test: no

ACCLIMATION
- Acclimation conditions (same as test or not): dult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 2) 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.


Test type:
static
Water media type:
freshwater
Remarks:
Elendt M7 Medium
Limit test:
no
Total exposure duration:
48 h
Hardness:
no data
Test temperature:
18-22 ± 1”C
pH:
The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC
Dissolved oxygen:
equal to or greater than 3 mg D.O./L
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Chemical analysis of the 10 and 100 mg/L test preparations at 0 hours showed measured test concentrations to range from 11 to 99 mg/L. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type (delete if not applicable): The test vessels were covered
- Aeration: no
- Material, size, headspace, fill volume: 150 mL glass beakers containing 100 mL Elendt M7 medium
- 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 (Elendt M7 medium) used for both the range-finding and definitive tests
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- light intensity: between 200 and 1200 Lux. The light intensity during the light period was measured using an ATP Instrumentation Lux meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.L.: 40 - 54 mgL
Details on results:
- Mortality of control: none
- Other adverse effects control: no
- Abnormal responses: none
- The No Observed Effect Concentrations after 24 and 48 hours exposure were 32 and 18 mg/L, respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 56 and 32 mg/L, respectively. The slopes and their standard errors of the response curves at 24 and 48 hours were 5.0 (SE = 0.04) and 7.1 (SE = 0.03) respectively.
- Sub-Lethal Effects: Sub lethal effects of exposure were observed in all test concentrations. These responses were trapping at the water surface and reduced mobility. No sub-lethal effects of exposure were observed in the control group.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Time Point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC (mg/L)
24 0.79 0.73 - 0.86 0.56 1.0
48 0.75 0.56 - 1.0 0.56 1.0
- 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:
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum Likelihood regression. The LOEC and the NOEC at 24 and 48 hours were calculated using the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal
Concentration
(mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

Nominal
Concentration
(mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

R1 – R4 = Replicates 1 to 4

N = Normal

Validity criteria fulfilled:
yes
Remarks:
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
Conclusions:
The acute toxicity of the test item to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. Based on nominal concentrations, the 48-h EC50 was determined to be 46 mg/L with a 95% confidence interval ranging from 40 to 54 mg/L. The results of the test can be considered reliable without restriction.

Description of key information

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.

Following a preliminary range‑finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of approximately 21 °C to 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

Analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 9.5 to 104 mg/L. Analysis of the old test media at 48 hours showed measured test concentrations to range from 11 to 103 mg/L. As all measured concentrations were within 20% of the nominal concentrations it was considered justifiable to base the results on the nominal test concentrations. Based on nominal concentrations, the 48-h EC50 was determined to be 46 mg/L with a 95% confidence interval ranging from 40 to 54 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
46 mg/L

Additional information