2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)prop-1-enyl]-1,3,3-trimethyl-3H-indolium acetate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data for the target chemical is summarized based on the structually and functionally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: refer below principle

Principles of method if other than guideline:

WoE study was prepared for toxicity of test chemical by considering WoE 2, 3 and 4

GLP compliance:

not specified

Analytical monitoring:

yes

Remarks:

2

Details on test solutions:

2: The test substance was soluble in Adams media. Therefore, the test solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media. Achieving test concentrations of 0.125 mg/L,0.25 mg/L,0.5 mg/L,1 mg/L, and 2 mg/L, respectively. Test water:ADaM’s media is used, prepared in MiliQ water.

3: The stock solution 20 mg/L was prepared by dissolving dark violet iquid in reconstituted water. 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:

2: - Common name: Water flea
- Source: Own breeding of daphnia magna
- Food: No feeding after hatching

3: 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:

other: 2: semi-static; 3: static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

2: 145.5 mg of CaCO3

Test temperature:

2: 20±2° C; 3: 20±1°C

pH:

2: 7.1; 3: Test at 0.100 mg/l: 8.1 (changed to 7.9 during test)
Control: 7.7 (Changed to 7.9 during test)

Dissolved oxygen:

3: higher than 8.7 mg/L at the end of test

Nominal and measured concentrations:

2: 0.5625 mg/L,1.125mg/L, 2.25mg/L, 4.5mg/L, 9mg/L; 3: 0, 0.006, 0.012, 0.025, 0.050, 0.100 mg/l

Details on test conditions:

2: TEST SYSTEM
- Test vessel: Glass beaker
- Aeration: No aeration during experiment
- Material, size, headspace, fill volume: 25 ml of glass beaker filled with 20 ml media having headspace of 5 ml
- No. of organisms per vessel: 10 daphnids

OTHER TEST CONDITIONS
- Photoperiod:16 hours light and 8 hours dark

3: 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:

3: Potassium dichromate (K2Cr2O7)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.037 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 0.025-0.054 mg/l

Remarks:

3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 4

Results with reference substance (positive control):

3: - Results with reference substance valid
- EC50: 0.76 mg/L (24 hours)

Reported statistics and error estimates:

3: EC50 was calculated using non linear regression by the software Prism 4.0

OBSERVATION:
1. Immobility
Nominal	Number	of	Immobility
Concentration	Daphnids	0 h	24 h	48 h
(mg/l)
Control	10	-	-	-
0.125	10	-	-	-
0.25	10	-	-	-
0.5	10	-	-	5
1	10	-	7	10
2	10	-	10	10

9.2. pH and oxygen content:
Nominal Concentration Number of		pH(mg/L)
(mg/l)	daphnids	0h	48h
Control	10	7.3	7.7
0.125	10	7.5	7.6
0.25	10	7.5	7.7
0.5	10	7.6	7.8
1	10	7.6	7.8
2	10	7.6	7.9
Nominal Concentration Number of		DO(mg/L)
(mg/l)	daphnids	0h	48h
Control	10	6.9	6.9
0.125	10	6.9	6.9
0.25	10	6.9	6.9
0.5	10	6.9	6.9
1	10	6.9	6.9
2	10	6.9	6.9
9.3. Test temperature
Nominal Concentration Number of		Temparature
(mg/l)	daphnids	0h	48h
Control	10	21.9	20.8
0.125	10	21.9	20.8
0.25	10	21.9	20.8
0.5	10	21.9	20.8
1	10	21.9	20.8
2	10	21.9	20.8

Validity criteria fulfilled:

yes

Remarks:

2: 1. In the control, including the control containing the solubilising agent, not more that 10 percent of the daphnids should have been immobilized..2. The dissolved oxygen concentration at the end of the test should be 3 mg/l in control and test vessels

Conclusions:

Study was conducted to determine effecttive concentration of test chemical on aquatic invertebrates for 48 h. The EC50 value for aquatic invertebrates for test chemical was determined to be ranges from 0.037-0.5 mg/L

Executive summary:

Data available for the structurally similar and closely related read across chemicals study has been reviewed to determine the short term toxicity of the test chemical on the growth of aquatic invertebrates. The studies are as mentioned below:

The first study of daphnia Immobilization Test was conducted in according to OECD Guideline 203 for reaction mass of test chemical . The test substance was soluble in Adams media. Therefore, the test solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media. Achieving test concentrations of 0.125 mg/L,0.25 mg/L,0.5 mg/L,1 mg/L, and 2 mg/L, respectively. Test water:ADaM’s media is used, prepared in MiliQ water and test Daphnia magna were exposed to these concentration for 48 hours. Test conducted under the semi-static system in which 25 ml of glass beaker filled with 20 ml media having headspace of 5 ml were used. Based on nominal concentrations, experimental median effective Concentrations [EC-50 (48 h)] for reaction mass of test chemical on test daphnia was determine to be 0.5 mg/L. Test consider to be valid as fulfills all the criteria as per standards, as not more that 10 percent of the daphnids were immobilized and the dissolved oxygen concentration at the end of the test was 3 mg/l in control and test vessels. Based on the EC50, it can be consider that the chemical was toxic and can be consider to be classified as aquatic acute 1 as per the CLP classification criteria.

Second determination of the inhibition of the mobility of daphnids study was carried out with the substance according to OECD Guideline 202. The test substance was tested at the different nominal concentrations 0, 0.006, 0.012, 0.025, 0.050, 0.100 mg/l. Effects on immobilisation were observed for 48 hours. EC50 was calculated using non linear regression by the software Prism 4.0. Potassium dichromate (K2Cr2O7) used as reference substance in study. The was carried out in 50 ml glass vessel of at temp of 20±1°C and 7.9 pH. The stock solution 20 mg/L was prepared by dissolving dark violet iquid in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.The median effective concentration (EC50) for the test substance in Daphnia magna was determined to be 0.037 mg/L for immobilisation effects. This value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified as aquatic acute 1 as per the CLP criteria.

The last short term toxicity to aquatic invertebrates study was performed in Daphnia magna for 48 hrs. After the experiment,the EC50 value for aquatic invertebrates for test chemical was determined to be 0.06 mg/l. Based on the value, test chemical was considered to be toxic in nature and can be classified in aquatic acute category 1 as per CLP regulation.

On the baisis of above observations, the EC50 value for aquatic invertebrates for test chemical was determined to be ranges from 0.037-0.5 mg/L. Based on the value, test chemical was considered to be toxic in nature and can be classified in aquatic acute category 1 as per CLP regulation.

Description of key information

Study was conducted to determine effecttive concentration of test chemical on aquatic invertebrates for 48 h. The EC50 value for aquatic invertebrates for test chemical was determined to be ranges from 0.037-0.5 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.5 mg/L

Additional information

Data available for the structurally similar and closely related read across chemicals study has been reviewed to determine the short term toxicity of the test chemical on the growth of aquatic invertebrates. The studies are as mentioned below:

The first study of daphnia Immobilization Test was conducted in according to OECD Guideline 203 for reaction mass of test chemical . The test substance was soluble in Adams media. Therefore, the test solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media. Achieving test concentrations of 0.125 mg/L,0.25 mg/L,0.5 mg/L,1 mg/L, and 2 mg/L, respectively. Test water:ADaM’s media is used, prepared in MiliQ water and test Daphnia magna were exposed to these concentration for 48 hours. Test conducted under the semi-static system in which 25 ml of glass beaker filled with 20 ml media having headspace of 5 ml were used. Based on nominal concentrations, experimental median effective Concentrations [EC-50 (48 h)] for reaction mass of test chemical on test daphnia was determine to be 0.5 mg/L. Test consider to be valid as fulfills all the criteria as per standards, as not more that 10 percent of the daphnids were immobilized and the dissolved oxygen concentration at the end of the test was 3 mg/l in control and test vessels. Based on the EC50, it can be consider that the chemical was toxic and can be consider to be classified as aquatic acute 1 as per the CLP classification criteria.

Second determination of the inhibition of the mobility of daphnids study was carried out with the substance according to OECD Guideline 202. The test substance was tested at the different nominal concentrations 0, 0.006, 0.012, 0.025, 0.050, 0.100 mg/l. Effects on immobilisation were observed for 48 hours. EC50 was calculated using non linear regression by the software Prism 4.0. Potassium dichromate (K2Cr2O7) used as reference substance in study. The was carried out in 50 ml glass vessel of at temp of 20±1°C and 7.9 pH. The stock solution 20 mg/L was prepared by dissolving dark violet iquid in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.The median effective concentration (EC50) for the test substance in Daphnia magna was determined to be 0.037 mg/L for immobilisation effects. This value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified as aquatic acute 1 as per the CLP criteria.

The last short term toxicity to aquatic invertebrates study was performed in Daphnia magna for 48 hrs. After the experiment,the EC50 value for aquatic invertebrates for test chemical was determined to be 0.06 mg/l. Based on the value, test chemical was considered to be toxic in nature and can be classified in aquatic acute category 1 as per CLP regulation.

On the baisis of above observations, the EC50 value for aquatic invertebrates for test chemical was determined to be ranges from 0.037-0.5 mg/L. Based on the value, test chemical was considered to be toxic in nature and can be classified in aquatic acute category 1 as per CLP regulation.