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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:
weight of evidence
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:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Remarks:
DMSO was used as a vehicle.
Details on test solutions:
The stock solution 200 g/l was prepared by dissolving white powder in DMSO. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted 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
sample at concentration 80 mg/l: pH = 7.7 changed to pH = 7.5 during the test
control: pH = 7.8 did not change during the test
Dissolved oxygen:
higher than 7.8 mg/L at the end of test both in the control and the sample
Nominal and measured concentrations:
Nominal test chemical concentration used for the study were 0, 5, 10, 20, 30, 40 and 80 mg/l, respectively.
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.:
26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % C. I. = 23.5 to 28.9 mg/l
Results with reference substance (positive control):
- Results with reference substance valid
- EC50: 0.79 mg/L (24 hours)

Reported statistics and error estimates:
EC50 was calculated using non linear regression by the software Prism 4.0.

In control solution containing reconstituted water without the test chemical & another control vessel containing reconsituted water, DMSO without the test chemical, no daphnids were immobilized at the end of the test.

Validity criteria fulfilled:
yes
Conclusions:
Based on the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr EC50 value was determined to be 26.0 mg/l (95 % C. I. - 23.5 to 28.9 mg/l).
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”. Daphnia magna was used as a test organism for the study. The stock solution 200 g/l was prepared by dissolving white powder in DMSO. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Nominal test chemical concentrations used for the study 0, 5, 10, 20, 30, 40 and 80 mg/l, respectively. Study was performed using 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. Control solution vessels containing reconstituted water without the test chemical and reconsituted water, DMSO without the test chemical were also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. In both the control vessels, no daphnids were immobilized at the end of the test. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 26 mg/l (95 % C. I. - 23.5 to 28.9 mg/l ). Thus, test chemical is considered as toxic to aquatic invertebrates at environmental related concentrations and considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Description of key information

The overall study concludes that target test chemical is toxic to aquatic environment with EC50 value after 48 hours exposure ranging from 4.04 to 52 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
52 mg/L

Additional information

Data available for target chemical and structurally & functionally similar read across analogues, has been reviewed to determine the effect caused on fresh water aquatic organisms. The studies are summarised below:

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”. Daphnia magna was used as a test organism for the study. The stock solution 200 g/l was prepared by dissolving white powder in DMSO. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Nominal test chemical concentrations used for the study 0, 5, 10, 20, 30, 40 and 80 mg/l, respectively. Study was performed using 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. Control solution vessels containing reconstituted water without the test chemical and reconsituted water, DMSO without the test chemical were also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. In both the control vessels, no daphnids were immobilized at the end of the test. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 26 mg/l (95 % C. I. - 23.5 to 28.9 mg/l ). Thus, test chemical is considered as toxic to aquatic invertebrates at environmental related concentrations and considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Another short term toxicity to aq. Invertebrate study was conducted for 48 hrs for assessing the effect of test chemical. The study was performed in reconstituted water in a static system at 17°C temperature.Daphnia pulex(Water flea) of less than 24 hr old was used as a test organism for the study.Test organism was fed with 1:1:1:1:4 mixture of four single species of green algae cultures with cerophyl medium (0.3 ng/ml). The resulting conc. of algae (cells/ml) in the daphnid culture water wereChlorella vulgaris(1,700 cells/ml);Chlorella pyrenoidosa(1,300 cells/ml);Ankistrodesmus falcatus(300 cells/ml); andChlamydomonas reinhardii(230 cells/ml). Feeding to the test organism was not done during the test. Total 5 concentrations of test chemical were taken for the study. Exact test chemical conc. was not known. The nominal concentrations of test chemical in each treatment, except the control and highest concentration treatment, were at least 60% of the next higher concentration and fit a geometric progression. Acetone was used as a vehicle. Each test vessel contains atleast 10 neonates/vessel in 150 ml of water. All experiments were performed in 4 replicates. These test vessels were kept at 17°C for an exposure period of 48 hrs. After a period of 48 hrs, immobilization of the test organism was evaluated. The data (percent immobilization at 48 h) were evaluated by probit analysis using a modified version of the IBM SSP package computer program.On the basis of effect on themobility of the test organismDaphnia pulex, the 48 hr EC50 value was determined to be 4.04 mg/l with standard error of ± 0.47.Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic invertebrates at environmental relevant concentrations and considered to be classified in ‘aquatic chronic category 2’ as per the CLP classification criteria.

For the test chemical, short term toxicity to aq. Invertebrate study was conducted for 48 hrs for assessing the effect of test chemical. The study was performed in accordance with the EU Method C.2 (Acute Toxicity for Daphnia) under static conditions. Test chemical concentration was verified analytically. Daphnia magna (Water flea) was used as a test organism. On the basis of effect on the mobility of the test organismDaphnia magna(Water flea), the 48 hr EC50 was determined to be 52 mg/l (measured concentration). Based on this value, test chemical can be considered as toxic to aquatic invertebrates and considered to be classified in ‘aquatic chronic category 3’ as per the CLP classification criteria.

Additional 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”. Daphnia magna was used as a test organism for the study. The stock solution 100 mg/l was prepared by dissolving white powder in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Nominal test chemical concentrations used for the study 0, 0, 1.2, 2.5, 5, 10, 20 and 40 mg/l, respectively. Study was performed using 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. Control solution vessels containing reconstituted water and reconsitituted water including acetone without the test chemical were also setup during the study. 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 run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 48.1 mg/l (95 % C. I. - 39.3 to 58.9 mg/l ). Thus, test chemical is considered as toxic to aquatic invertebrates at environmental related concentrations and considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

On the basis of the review from expert judgements and considering the study results conducted as per the OECD guideline 202 for the target chemical by applying the weight of evidence approach, it is concluded that the test chemical can be consideredastoxic to aquatic invertebrates at environmental related concentrations and considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.