[No public or meaningful name is available]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2020 to 21 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Range-finding Test
A sample of each test concentration was taken and stored frozen at 0 hours prior to analysis with the samples taken at 48 hours in order to determine the stability of the test item under test conditions. A duplicate set of samples was taken at 48 hours and stored frozen for further analysis if required, however due to a technical oversight no duplicate set of samples was taken at 0 hours.

- Definitive Test
Samples were taken from the control and the 100% v/v saturated solution test group from the fresh bulk test preparation at 0 and 24 hours and from the old/expired pooled replicates at 24 and 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate sets of samples were taken at 0, 24 (fresh and old) and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation. Therefore, a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test material concentration of approximately 0.026 mg/L could be obtained using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL discarded).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Justification for species: recommended by the study guidelines and are commonly used in acute freshwater invertebrate toxicity tests
- Age at study initiation: <24 hours old; 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. Daphnids used for testing where not first brood progeny.
- Source: Test facility culture
- Feeding during test: no

CULTURE AND CULTURE CONDITIONS
- Culture vessel: 150 mL glass beaker containing 100 mL of reconstituted water (ISO Medium)
- Food type: Algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension.
- Feeding frequency: Each culture was fed daily
- Water Type: Reconstituted water ISO medium (Ingredients: CaCl2.2H2O 294 mg/L, MgSO4.7H2O 123 mg/L, NaHCO3 65 mg/L, KCl 5.8 mg/L) pH: 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air saturation value

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

19 to 20 °C

pH:

7.7 to 8.1

Dissolved oxygen:

8.6 to 9.0 mg O2/L

Nominal and measured concentrations:

Range-finding study: 0.10, 1.0,10,100% v/v Saturated Solution (nominal)

Definitive study: 100% v/v saturated solution (nominal), equivalent to 0.014 mg/L (geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessels
- Fill volume: 100 mL
- Type: closed
- Aeration: no.
- 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 ISO medium (Ingredients: CaCl2.2H2O 294 mg/L, MgSO4.7H2O 123 mg/L, NaHCO3 65 mg/L, KCl 5.8 mg/L) pH: 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air saturation value.
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment of the pH of the test media to 6.0 was deemed necessary in order to aid solubility/stability of the test material.
- Photoperiod: 16 hours light: 8 hours dark with a 20 minute transition periods.
- Light intensity: 819 and 91400 Lux

EFFECT PARAMETERS MEASURED
Any immobilisation 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 immobilised if they were unable to swim within 15 seconds after gentle agitation.

WATER QUALITY MEASUREMENTS
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal and on termination of the test after 48 hours represent those of the used or 24 hour old test preparations. The pH and dissolved oxygen concentration were measured using a calibrated Hach Flexi handheld meter whilst the temperature was measured using a calibrated 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.

RANGE-FINDING STUDY
- Test concentrations: 0.10,1.0,10 and 100% v/v of the saturated solution
- Results used to determine the conditions for the definitive study: yes

A nominal amount of test material (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 500 mL used to pre condition the filter was discarded), to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range finding test, five daphnids were placed in each of 2 replicate test and control vessels 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 and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 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 material.

INITIAL EXPERIMENT
Based on the results of the range-finding test a “limit test” was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration no significant immobilization was observed. At the 24-Hour observation period it was noticed that greater than 10% of the controls were recorded as immobilized or other signs of distress and therefore the test was terminated and repeated. The results from this study were not used for reporting purposes.

DEFINITIVE STUDY
Based on the results of the range finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration no significant immobilisation was observed.
A nominal amount of test material (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 500 mL used to pre condition the filter was discarded), to give a 100% v/v saturated solution.
The single limit concentration was inverted several times to ensure adequate mixing and homogeneity.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.014 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

> DEFINITIVE TEST
- VERFICATION OF TEST CONCENTRATIONS
Analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations at 0.014 and 0.014 mg/L, respectively as test material. The chemical analysis of the old/expired test preparations at 24 and 48 hours showed measured test concentrations to range from 0.014 and 0.012 mg/L as test material. Given that there was a slight decline in the measured concentrations at 24 and 48 hours it was considered justifiable to base the results on the geometric mean measured test concentration. The geometric mean measured test concentration was determined to be 0.014 mg/L as test material.

- IMMOBILISATION DATA
There was no immobilization in 20 daphnids exposed to a geometric mean measured test concentration of 0.014 mg/L as test material for a period of 48 hours. However, there was one immobilised daphnid observed in the control after 48 hours. The NOEC after 24 and 48 hours exposure was determined to be ≥0.014 mg/L as test material. The Lowest Observed Effect Concentrations (LOEC) after 24 and 48 hours exposure could not be determined.

- SUB-LETHAL EFFECTS:
No sub lethal effects of exposure were observed throughout the test.

- VALIDITY CRITERIA:
The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress (actual 5%), 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 (actual ≥8.6 mg/L).

- WATER QUALITY CRITERIA
Temperature was maintained at 19 °C to 20 °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 819 to 914 Lux.

- TEST MATERIAL SOLUBILITY
At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.

Results with reference substance (positive control):

Daphnids were exposed to potassium dichromate at concentrations of of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and was conducted from 23 January 2020 to 07 February 2020. The EC50 at 24 hours was 0.90 mg/L with a 95% confidence interval of 0.80 to 1 mg/L and at 48 hours exposure was 0.79 mg/L with a 95% confidence interval of 0.71 to 0.87. The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

An estimate of the EC50 values was given by inspection of the immobilisation data.

Range-finding tests:

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 immobilisation was observed.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the Limit of Quantification (LOQ) to 0.014 mg/L as test material. Analysis of the corresponding aged preparations at 48 hours showed measured test concentrations to range from less than the LOQ to 0.0087 mg/L as test material. There was a significant decline in the measured concentrations at 48 hours indicating that the test material was not stable under test conditions.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, exposure of Daphnia magna to the test material gave a 48-hour EC50 value based on the geometric mean measured test concentration of > 0.014 mg/L as test material. The 48-hour No Observed Effect Concentration (NOEC) was determined to be ≥ 0.014 mg/L as test material. This study showed that there were no toxic effects at saturation/limit of water solubility.

Executive summary:

The short-term toxicity of the test material to aquatic invertebrates was investigated in a study which was conducted in accordance with the standardised guideline OECD 202, EU Method C.2 and under GLP conditions.

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

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.026 mg/L was obtained from a 50 mL saturated solution of test material stirred for 24 hours at regular pH, indicating this to be the limit of water solubility of this material under test conditions.  Preliminary testing confirmed that different pH levels and stirring times had no significant impact on the concentration of test material dissolved, hence 24 hours stirring and regular pH levels were used for testing.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to a single aqueous solution of the test material at a limit concentration of 100% v/v saturated solution, for 48 hours at a temperature of 19 °C to 20 °C under semi-static test conditions.  The test material solution was prepared by stirring an excess (50 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours.  After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 500 mL used to pre-condition the filter was discarded), to give a 100% v/v saturated solution of the test material. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations at 0.014 and 0.014 mg/L, respectively as test material. The chemical analysis of the old/expired test preparations at 24 and 48 hours showed measured test concentrations to range from 0.014 and 0.012 mg/L as test material. Given that there was a slight decline in the measured concentrations at 24 and 48 hours the results are therefore based on the geometric mean measured test concentration. The geometric mean measured test concentration was determined to be 0.014 mg/L as test material.

There was no immobilization in 20 daphnids exposed to a geometric mean measured test concentration of 0.014 mg/L as test material for a period of 48 hours and no sub lethal effects of exposure were observed throughout the test.

The 48-hour EC50 value based on the geometric mean measured test concentration was therefore considered to be > 0.014 mg/L as test material. This study showed that there were no toxic effects at saturation/limit of water solubility.

Description of key information

Short-term toxicity to aquatic invertebrates:

Under the conditions of this study, exposure of Daphnia magna to the test material gave a 48-hour EC50 value based on the geometric mean measured test concentration of > 0.014 mg/L as test material. The 48-hour No Observed Effect Concentration (NOEC) was determined to be ≥ 0.014 mg/L as test material. This study showed that there were no toxic effects at saturation/limit of water solubility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 0.014 mg/L

Additional information

Short-term toxicity to aquatic invertebrates:

The short-term toxicity of the test material to aquatic invertebrates was investigated in a study which was conducted in accordance with the standardised guideline OECD 202, EU Method C.2 and under GLP conditions.

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

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.026 mg/L was obtained from a 50 mL saturated solution of test material stirred for 24 hours at regular pH, indicating this to be the limit of water solubility of this material under test conditions.  Preliminary testing confirmed that different pH levels and stirring times had no significant impact on the concentration of test material dissolved, hence 24 hours stirring and regular pH levels were used for testing.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to a single aqueous solution of the test material at a limit concentration of 100% v/v saturated solution, for 48 hours at a temperature of 19 °C to 20 °C under semi-static test conditions.  The test material solution was prepared by stirring an excess (50 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours.  After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 500 mL used to pre-condition the filter was discarded), to give a 100% v/v saturated solution of the test material. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations at 0.014 and 0.014 mg/L, respectively as test material. The chemical analysis of the old/expired test preparations at 24 and 48 hours showed measured test concentrations to range from 0.014 and 0.012 mg/L as test material. Given that there was a slight decline in the measured concentrations at 24 and 48 hours the results are therefore based on the geometric mean measured test concentration. The geometric mean measured test concentration was determined to be 0.014 mg/L as test material.

There was no immobilization in 20 daphnids exposed to a geometric mean measured test concentration of 0.014 mg/L as test material for a period of 48 hours and no sub lethal effects of exposure were observed throughout the test.

The 48-hour EC50 value based on the geometric mean measured test concentration was therefore considered to be > 0.014 mg/L as test material. This study showed that there were no toxic effects at saturation/limit of water solubility.