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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:
2017-08-02 to 2017-10-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EC No 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D26-3-3-1
- Expiration date of the lot/batch: 2018-12-01
- Purity: 47.4 wt%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

OTHER SPECIFICS:
- Physical state/Appearance: Brown viscous liquid
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 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.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water-flea
- Source: The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
- Age at study initiation: 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.
- Feeding during cultivation: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding during test: no

ACCLIMATION
- Acclimation period: not relevant
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
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/L
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Final test:
Nominal concentrations: 100 mg/L
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to be 102% nominal concentration. 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: glass jars
- Type (delete if not applicable): covered to reduce evaporation
- Material, size, headspace, fill volume: 150 mL glass jars containing approximately 100 mL of test preparation were used
- Aeration: not during the test
- 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: Elendt M7 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement:
* 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 Instrumentation Lux meter.
* The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: between 200 and 1200 Lux

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, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

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:
Potassium dichromate
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:
- Mortality of control: none
- Other adverse effects control: no
- Effect concentrations exceeding solubility of substance in test medium: 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):
- Results with reference substance valid? Yes
- 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, however, throughout the positive control the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test. The results from the positive control with potassium dichromate were within the normal range for this reference item
Reported statistics and error estimates:
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: 48-h EC50 = 1.2 mg/L (95% C.L. 1.1 - 1.3 mg/L)

Table 2: 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 the freshwater invertebrate Daphnia magna has been investigated in a 48 hour static test according to OECD guideline 202. Based on analytically confirmed nominal concentrations, the 48-h EC50 was determined to be greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L. The results of the test can be considered reliable without restriction.
Executive summary:

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 a nominal concentration of 100 mg/L for 48 hours at a temperature of approximately 22°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 88% to 93% of nominal and so the results are based on nominal test concentrations only.

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.It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

The study of the laboratories ENVIGO (2018), investigating the acute toxicity of the test substance in Daphnia magna according to OECD guideline 202, was considered as the key study for endpoint coverage. 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 nominal concentration of 100 mg/L for 48 hours at a temperature of approximately 22°C under static test conditions (limit test). Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

An EC50 >100 mg/L (based on analytically confirmed nominal test concentrations) was found after an exposure of 48 hours in a static test in freshwater. Based on the results it can be concluded that the test substance is not harmful to aquatic invertebrates.

Key value for chemical safety assessment

Additional information