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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:
Experimental start date - 09 October 2020; Experimental completion date - 02 December 2020
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: 4-Hydroxycoumarin
CAS Number: 1076-38-6
Batch: HCN20002
Purity: >98%
Physical State/Appearance: Beige powder
Expiry Date: 26 September 2022
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and the 100 % v/v saturated solution test groups from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were taken for immediate analysis. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Reconstituted water (Elendt M4 medium) used for both the range-finding and definitive tests.

Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

A nominal amount of test item (1100 mg) was dispersed in 11 L of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and the preparation filtered as a precautionary measure through a 0.2 μm Gelman Acrocap filter (the first approximate 100 mL used to pre-condition the filter was discarded), to give a 100% v/v saturated solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures. Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 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 (Raphidocelis subcapitata) and GEMMA Micro 300 fish 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
Hardness:
2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
19 °C to 21 °C
pH:
6.6 - 8.0
Dissolved oxygen:
8.7 - 9.3
Nominal and measured concentrations:
Range finding test - 0.1, 1, 10, 100% v/v saturated solution.
Definitive test - 100 mg/L
Details on test conditions:
As in the range-finding test 150 mL glass vessels containing approximately 100 mL of test preparation were used. At the start of the test five 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 and 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.

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 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution. No sub-lethal effects of exposure were observed throughout the test.
Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at highest attainable test concentration, no significant immobilization was observed.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
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:
Verification of Test Concentrations: Analysis of the test preparations at 0 hours showed the measured test concentration to be 100 mg/L. There was no significant change in the measured concentration at 48 hours as the measured concentration was determined to be 100 mg/L and so the results are based on measured test concentration.

Immobilization data: There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours.
Sub-Lethal Effects: No sub-lethal effects of exposure were observed throughout the test.
Results with reference substance (positive control):
The 48h EC50 for positive control was found to be 0.79 mg/L. The corresponding NOEC and LOEC was found to be 0.56 mg/L and 1.0 mg/L respectively.

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 19 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration. The pH in the test concentration was observed to be 6.6 compared to a control pH of 8.0. Despite the pH in the test concentration being lower than the control, as it was still within the range required for the test this was deemed acceptable.

Throughout the test the light intensity was observed to be in the range 944 to 956 Lux.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Definitive test:

 Measured 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

5N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

 

 Measured 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

Water Quality Measurements

Measured Concentration (mg/L)

0 Hours

24 Hours

48 Hours

pH

mg O2/L

Temperature

°C

Temperature ºC

pH

mg O2/L

Temperature

°C

Control

R1

8.0

9.0

20

20

8.1

9.3

19

100

R1

6.6

8.7

21

20

7.9

9.0

19

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the measured test concentration gave a 48 hour EC50 value of greater than 100 mg/L. The NOEC was 100 mg/L.
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.

 

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that the test item was soluble with the aid of prolonged stirring and therefore was prepared as a saturated solution.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of 19 °C to 21 °C under static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period as a precautionary measure the preparation was filtered through a 0.2 µm Gelman Acrocap filter (the first approximate 100 mL used to pre-condition the filter was discarded) to give a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparation at 0 hours showed the measured test concentration to be 100 mg/L. There was no significant change in the measured concentration at 48 hours as the measured concentration was determined to be 100 mg/L and so the results are based on the measured testconcentration.

Exposure of Daphnia magna to the test item gave EC50 values based on the nominal test concentration of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the measured test concentration gave a 48 hour EC50 value of greater than 100 mg/L. The NOEC was 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional 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.

 

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that the test item was soluble with the aid of prolonged stirring and therefore was prepared as a saturated solution.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of 19°C to 21°C under static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period as a precautionary measure the preparation was filtered through a 0.2 µm Gelman Acrocap filter (the first approximate 100 mL used to pre-condition the filter was discarded) to give a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparation at 0 hours showed the measured test concentration to be 100 mg/L. There was no significant change in the measured concentration at 48 hours as the measured concentration was determined to be 100 mg/L and so the results are based on the measured testconcentration.

Exposure of Daphnia magna to the test item gave EC50 values based on the nominal test concentration of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.