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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 test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Principles of method if other than guideline:
Daphnia sp. acute immobilisation test was carried out on Daphnia magna with the test substance according to OECD Guideline 202.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Acetone
Details on test solutions:
The stock solution used for the exposure assessment was prepared by dissolving colourless dense liquid in acetone.
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

ACCLIMATION - No data available
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
+/- 1 hour
Post exposure observation period:
no
Test temperature:
20+/-1 C
pH:
7.8 did not change during test
Dissolved oxygen:
7.8 mg/l
Nominal and measured concentrations:
nominal: 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: No - 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.:
2.59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CI was 1.81 - 3.70 mg/l
Results with reference substance (positive control):
4-hour-EC50 K2Cr2O7= 0.79 mg/L.
Reported statistics and error estimates:
EC50 was calculated using non linear regression by the software Prism 4.0
Validity criteria fulfilled:
yes
Conclusions:
Based on the immobility of daphnia magna by the chemical exposure for 48 hrs, the EC50 was determine to be 2.59 mg/l.
Executive summary:

An acute immobilisation test was used to test how a range of concentrations of test chemical exerts different degrees of toxic effects on the swimming capability of Daphnia magna under otherwise identical test conditions. The test was performed in close resemblance to OECD guideline 202. The testing aim was to determine a EC50 after 48 hours of exposure to D. magna. The stock solution used for the exposure assessment was prepared by dissolving colourless dense liquid in acetone. Daphnids were exposed to chemical in 50 ml beakers in a volume of 25 ml of liquid solution containing both the chemical and media as specified in OECD 202. The beakers were placed in a temperature controlled room at 20±1 degrees Celsius. The D. magna (age ≤24) used for the test. The animals were exposed to stock solution prepared by adding dense colourless liiquid in acetone. There were 5 Daphnia per test vessels and 4 replicates per concentration. The positive control/reference substance used in the teste showed an expected result and gave a EC50 that corresponded to previous exposures with this chemical in D. magna. Immobilisation effects in D. mangaby chemical exposure was evident after 48 hours in exposure concentrations above 1 mg/L (i.e. EC20). The EC50 was defined as a concentration that immobilizes 50% of the exposed D. magna. The % of immobilization in D. magna after 48 hours of exposure to test chemical were used in a nonlinear regression by Graphpad Software Prism 4.0 (San Diego, US). Based on the immobility of daphnia magna by the chemical exposure for 48 hrs, the EC50 was determine to be 2.59 mg/l (95% C.I: 1.81-3.7 mg/L). Thus, based on this value, test chemical can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 2 as per the CLP classification criteria.

Description of key information

An acute immobilisation test was used to test how a range of concentrations of test chemical exerts different degrees of toxic effects on the swimming capability ofDaphnia magnaunder otherwise identical test conditions. The test was performed in close resemblance to OECD guideline 202. The testing aim was to determine a EC50 after 48 hours of exposure toD. magna. The stock solution used for the exposure assessment was prepared by dissolving colourless dense liquid in acetone.Daphnidswere exposed to chemical in 50 ml beakers in a volume of 25 ml of liquid solution containing both the chemical and media as specified in OECD 202. The beakers were placed in a temperature controlled room at 20±1 degrees Celsius. TheD. magna(age ≤24) used for the test. The animals were exposed to stock solution prepared by adding dense colourless liiquid in acetone. There were 5Daphniaper test vessels and 4 replicates per concentration. The positive control/reference substance used in the teste showed an expected result and gave a EC50 that corresponded to previous exposures with this chemical inD. magna. Immobilisation effects inD. mangaby chemical exposure was evident after 48 hours in exposure concentrations above 1 mg/L (i.e. EC20). The EC50 was defined as a concentration that immobilizes 50% of the exposedD. magna. The % of immobilization inD. magnaafter 48 hours of exposure to test chemical were used in a nonlinear regression by Graphpad Software Prism 4.0 (San Diego, US). Based on the immobility of daphnia magna by the chemical exposure for 48 hrs, the EC50 was determine to be 2.59 mg/l (95% C.I: 1.81-3.7 mg/L).Thus, based on this value, test chemical can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 2 as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.59 mg/L

Additional information

An acute immobilisation test was used to test how a range of concentrations of test chemical exerts different degrees of toxic effects on the swimming capability of Daphnia magna under otherwise identical test conditions. The test was performed in close resemblance to OECD guideline 202. The testing aim was to determine a EC50 after 48 hours of exposure toD. magna. The stock solution used for the exposure assessment was prepared by dissolving colourless dense liquid in acetone.Daphnidswere exposed to chemical in 50 ml beakers in a volume of 25 ml of liquid solution containing both the chemical and media as specified in OECD 202. The beakers were placed in a temperature controlled room at 20±1 degrees Celsius. TheD. magna(age ≤24) used for the test. The animals were exposed to stock solution prepared by adding dense colourless liiquid in acetone. There were 5Daphniaper test vessels and 4 replicates per concentration. The positive control/reference substance used in the teste showed an expected result and gave a EC50 that corresponded to previous exposures with this chemical inD. magna. Immobilisation effects inD. mangaby chemical exposure was evident after 48 hours in exposure concentrations above 1 mg/L (i.e. EC20). The EC50 was defined as a concentration that immobilizes 50% of the exposedD. magna. The % of immobilization inD. magnaafter 48 hours of exposure to test chemical were used in a nonlinear regression by Graphpad Software Prism 4.0 (San Diego, US). Based on the immobility of daphnia magna by the chemical exposure for 48 hrs, the EC50 was determine to be 2.59 mg/l (95% C.I: 1.81-3.7 mg/L).Thus, based on this value, test chemical can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 2 as per the CLP classification criteria.