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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard OECD test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Principles of method if other than guideline:
Short term toxicity of test chemical to Daphnia magna was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Acetone was used as a vehicle.
Details on test solutions:
The stock solution 10 g/ml was prepared by dissolving white powder in acetone. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Source: Own breeding at University of Chemistry and Technology, Prague
- Age at study initiation (mean and range, SD): The animals used for the test shall be less than 24 h old and should not be first brood progeny
- Feeding during test: No feeding
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
± 1 hr
Test temperature:
20±1°C
pH:
without adjustment
the sample at concentration 16 mg/l: pH 7.3 changed to pH 7.7 during test
control: pH = 7.8 changed to pH = 7.7 during the test
control + acetone: pH = 7.8 changed to pH = 7.9 during the test
Dissolved oxygen:
higher than 7.6 mg/L at the end of test both in the control and the sample
Nominal and measured concentrations:
Nominal test chemical conc. used for the were 0, 0, 1, 2, 4, 8 and 16 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 ml glass vessel
- fill volume: 25 ml
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Natural water (surface or ground water), reconstituted water or dechlorinated tap water are acceptable as culturing and dilution water if D. magna survives in it for the duration of the culturing, acclimation and testing without showing signs of stress. Waters in the range pH 6 to pH 9, with hardness between 140 mg/l and 275 mg/l (as CaCO3) are recommended.
As an example, the preparation of dilution water meeting the requirements is described below.
Dissolve known quantities of reagents in water. The dilution water prepared shall have a pH of 7.8 ± 0.5, a hardness of (225 ± 50) mg/l (expressed as CaCO3), a molar Ca + Mg ratio close to 4 + 1 and a dissolved oxygen concentration above 7 mg/l.

Prepare the solutions specified below:
- Calcium chloride solution: Dissolve 117.6 g of calcium chloride dihydrate (CaCl2.2H2O) in water (4.2) and make up to 1 l with water (4.2).
- Magnesium sulfate solution: Dissolve 49.3 g of magnesium sulfate heptahydrate (MgSO4.7H2O) in water (4.2) and make up to 1 l with water (4.2).
- Sodium bicarbonate solution: Dissolve 25.9 g of sodium bicarbonate (NaHCO3) in water (4.2) and make up to 1 l with water (4.2).
- Potassium chloride solution: Dissolve 2.3 g of potassium chloride (KCI) in water (4.2) and make up to 1 l with water (4.2).

Mixing
Mix 2.5 ml of each of the four solutions and make up to 1 l with water.
The dilution water shall be aerated until the dissolved oxygen concentration has reached saturation and the pH has stabilized. If necessary, adjust the pH to 7.8 ± 0.5 by adding sodium hydroxide (NaOH) solution or hydrochloric acid (HCI). The dilution water prepared in this way shall not be further aerated before use.

- Sodium hydroxide solution, e.g. [NaOH] : 1 mol/l.
- Hydrochloric acid, e.g. [HCl] : 1 mol/l.

Reference substance:
Dissolve 600 mg of potassium dichromate (K2Cr2O7) in water and make up to 1 l with water (4.2).

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment done
- Photoperiod: Darkness
- Light intensity:

CALCULATION:
EC50 was calculated using non linear regression by the software Prism 4.0
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
6.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 5.6 to 8.4 mg/l
Results with reference substance (positive control):
- Results with reference substance valid
- EC50: 0.73 mg/L (24 hours)
Reported statistics and error estimates:
EC50 was calculated using non linear regression by the software Prism 4.0
Validity criteria fulfilled:
yes
Conclusions:
The median effective concentration (EC50) for the test chemical in Daphnia magna was determined to be 6.9 mg/L on the basis of mobility inhibition effects in a 48 hour study.
Executive summary:

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Own breeding stock at University of Chemistry and Technology, Prague of Daphnia magna was used as a test organism for the study. The stock solution 10 g/ml was prepared by dissolving white powder in acetone. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water. Nominal test chemical conc. used for the study were 0, 0, 1, 2, 4, 8 and 16 mg/L, respectively. Study was performed using total 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. On the basis of the effect of test chemical on the mobility of the test organism Daphnia magna, the 48 hr EC50 value was determined to be 6.9 mg/l with a 95% confidence interval value ranging from 5.6 to 8.4 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic invertebrates and thus can be considered to be classified in ‘aquatic chronic category 2’ as per CLP classification criteria.

Description of key information

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna(Experimental study report, 2018). The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Own breeding stock at University of Chemistry and Technology, Prague of Daphnia magna was used as a test organism for the study. The stock solution 10 g/ml was prepared by dissolving white powder in acetone. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water. Nominal test chemical conc. used for the study were 0, 0, 1, 2, 4, 8 and 16 mg/L, respectively. Study was performed using total 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. On the basis of the effect of test chemical on the mobility of the test organism Daphnia magna, the 48 hr EC50 value was determined to be 6.9 mg/l with a 95% confidence interval value ranging from 5.6 to 8.4 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic invertebrates and thus can be considered to be classified in ‘aquatic chronic category 2’ as per CLP classification criteria.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
6.9 mg/L

Additional information

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna(Experimental study report, 2018). The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Own breeding stock at University of Chemistry and Technology, Prague of Daphnia magna was used as a test organism for the study. The stock solution 10 g/ml was prepared by dissolving white powder in acetone. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water. Nominal test chemical conc. used for the study were 0, 0, 1, 2, 4, 8 and 16 mg/L, respectively. Study was performed using total 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. On the basis of the effect of test chemical on the mobility of the test organism Daphnia magna, the 48 hr EC50 value was determined to be 6.9 mg/l with a 95% confidence interval value ranging from 5.6 to 8.4 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic invertebrates and thus can be considered to be classified in ‘aquatic chronic category 2’ as per CLP classification criteria.