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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:
2019-08-06 to 2019-09-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 48 hours) by pooling the contents of the test beakers of each treatment.
All samples remained undiluted until sample preparation which was performed directly after sampling.
All samples were prepared stand-by immediately after sampling by liquid-liquid extraction with the organic solvent Cyclohexane. An aliquot of the resulting organic solvent extracts of the samples taken at test start were stored in a refrigerator (4 ± 4°C) until analysis, which was performed at the same day of the sampling of the test end samples.
Vehicle:
no
Details on test solutions:
Test Concentrations:
Due to the low water solubility limit of the test item a filtrate of a supersaturated stock suspension of nominal 100 mg/L and dilutions of thereof of 1:2, 1:4, 1:8 and 1:16 and a control were tested. The highest concentration is corresponding to following arithmetic mean measured concentrations of 0.258 µg test item/L.
Control:
In the control, test water was used without addition of the test item.
Dosage of Test Item:
The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on the daphnids, no concentrations above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 108.8 mg test item in 1088 mL test water. The stock suspension was stirred for 24 hours at room temperature to dissolve as much test item as possible. Afterwards, the solution was allowed to settle for 1 hour. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.2 µm cellulose acetate filter). The solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:2, 1:4, 1:8 and 1:16 dilutions. The test media were prepared just before introduction of the daphnids (= start of the test).
Appearance of the Test Item in Test Medium: There were no remarkable observations.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 0.50 to 19.50 hours old
Origin: The daphnids introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Reference Item: For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate
Test temperature:
20-21 °C at test start;
20 °C at test end
pH:
7.7 to 8.0 at test start;
7.7 to 7.8 at test end; and thus the pH-value did not vary by more than 1.5 units.
Dissolved oxygen:
7.1 to 8.9 mg/L at test start; 8.9 mg/L at test end
Nominal and measured concentrations:
Due to the low water solubility limit of the test item a filtrate of a supersaturated stock suspension of nominal 100 mg/L and dilutions of thereof of 1:2, 1:4, 1:8 and 1:16 and a control were tested.
The highest concentration is corresponding to following arithmetic mean measured concentrations of 0.258 µg test item/L.
Details on test conditions:
Test Environment: Controlled environment room
Measurement of pH, Dissolved Oxygen and Water Temperature:
The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature: 20.2 to 21.0 °C at test start; 20.0 to 20.2 °C at test end pH-Values: 7.7 to 8.0 at test start; 7.7 to 7.8 at test end
Dissolved Oxygen Concentration: 7.1 to 8.9 mg/L at test start; 8.9 mg/L at test end Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 650 to 950 lux (measured once during the test).
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.
Reference substance (positive control):
yes
Remarks:
For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
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
Remarks on result:
other: 100 mg test item/L is the nominal loading rate, a filtrate of 100 mg test item/L was tested, the mean measured concentration was 0.258 µg test item/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 100 mg test item/L is the nominal loading rate, a filtrate of 100 mg test item/L was tested, the mean measured concentration was 0.258 µg test item/L
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 100 mg test item/L is the nominal loading rate, a filtrate of 100 mg test item/L was tested, the mean measured concentration was 0.258 µg test item/L
Details on results:
After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the filtrate of 100 mg test item/L (>0.258 µg test item/L based on the arithmetic mean measured concentration).
Results with reference substance (positive control):
In the most recent test with the reference item potassium dichromate the EC50 after 24 hours was determined to be 0.932 mg test item/L, indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
Reported statistics and error estimates:
No statistical analysis was performed.
The EC50 could not be quantified due to the absence of toxicity of the test item and were determined to be higher than the highest test concentration. The NOEC and the LOEC were determined directly from the raw data.
Validity criteria fulfilled:
yes
Remarks:
Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.9 mg O2/L in the control and test vessels at the end of the test.
Conclusions:
The toxic effect of the test item to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be the filtrate of 100 mg test item/L (0.258 µg test item/L). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines.
The 48-hour LOEC and the 48-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L). These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L), the concentration representing the limit of solubility.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to arithmetic mean measured concentrations since the concentrations of the test item were not within ± 20% of the measured initial concentrations during the test. In addition, all reported results are given based on nominal concentrations (loading rate).
This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.
Executive summary:

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a static test to various concentrations under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Due to the low water solubility limit of the test item a filtrate of a supersaturated stock suspension of nominal 100 mg/L and dilutions of thereof of 1:2, 1:4, 1:8 and 1:16 and a control were tested. The highest concentration is corresponding to following arithmetic mean measured concentrations of 0.258 µg test item/L.

Biological Test Results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the filtrate of 100 mg test item/L (>0.258 µg test item/L based on the arithmetic mean measured concentration).

The 48-hour NOEC was determined to be the filtrate of 100 mg test item/L (0.258 µg test item/L). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines.

The 48-hour LOEC and the 48-hour EC50were clearly higher than the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L). These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L), the concentration representing the limit of solubility.

Analytical test results:

The quantification of the test item in the test samples was performed using liquid-liquid extraction with Cyclohexane followed by analysis via Gas Chromatography with MS/MS detection.

The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times (0 and 48h hours). The arithmetic mean measured values are :

0.258 µg test item/L in the filtrate of nominal 100 mg test item/L

<LOQ in the 1:2 dilution of filtrate

<LOQ in the 1:4 dilution of filtrate

<LOQ in the 1:8 dilution of filtrate

<LOD in the 1:16 dilution of filtrate

Conclusion:

The toxic effect of the test item to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be the filtrate of 100 mg test item/L (0.258 µg test item/L). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines.

The 48-hour LOEC and the 48-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L). These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L), the concentration representing the limit of solubility.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to arithmetic mean measured concentrations since the concentrations of the test item were not within ± 20% of the measured initial concentrations during the test. In addition, all reported results are given based on nominal concentrations (loading rate).

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Description of key information

The EC50 could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L), the concentration representing the limit of solubility.

GLP OECD 202: EC50 > 100 mg/L (nominal) corresponding to the mean measured concentration of 0.258 µg/L

Key value for chemical safety assessment

Additional information

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a static test to various concentrations under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Due to the low water solubility limit of the test item a filtrate of a supersaturated stock suspension of nominal 100 mg/L and dilutions of thereof of 1:2, 1:4, 1:8 and 1:16 and a control were tested. The highest concentration is corresponding to following arithmetic mean measured concentrations of 0.258 µg test item/L.

Conclusion:

The toxic effect of the test item to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be the filtrate of 100 mg test item/L (0.258 µg test item/L). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines.

The 48-hour LOEC and the 48-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L). These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L (>0.258 µg test item/L), the concentration representing the limit of solubility.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to arithmetic mean measured concentrations since the concentrations of the test item were not within ± 20% of the measured initial concentrations during the test. In addition, all reported results are given based on nominal concentrations (loading rate).