2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jan 2017 to 1 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Basic Red 14 sulfate
- Appearance: violet to sparkling dark blue solid
- Batch No.: 4030158820
- Date of manufacture: 31 March 2014
- Expiry date: March 2018
- Substance number: 16/0122-1
- Purity: 100 weight% main component
- Water solubility: 74.1 g/L ± 0.4 g/L at 20.0°C ± 0.5°C
- Storage condition of test material: at ambient temperature without exposure to light in a tightly sealed container
- Stability under test conditions: not specified

Analytical monitoring:

yes

Details on sampling:

Samples of all test item concentrations and the control at exposure initiation and at exposure termination were chemically analyzed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item in the amount of 10.2 mg was weighed in a glass crystallizer and quantitatively transferred into a flask by multiple washing with the Elendt M7 medium and filled up to 1020 mL. The test item concentration of 10 mg/L was visually homogeneous. Lower test item concentrations were prepared by sequential dilutions with the Elendt M7 medium in a ratio 1:1 (v/v) (i.e. 500 mL of higher test item concentration was mixed with 500 mL of the Elendt M7 medium).
- Controls: Elendt M7 medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: The standard laboratory culture cultivated at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology
- Culturing conditions: glass beakers with a capacity of 150 mL (one parent per vessel) containing 100 mL of Elendt M7 medium at room temperature with daily cycle 16 h light : 8 h dark (KANLUX electronic time programmer, Poland).
- Culture medium: The Elendt M7 medium was prepared on the basis of deionised water by adding stock solutions of reagent-grade chemicals. The stock solutions for culturing Daphnia magna were renewed on a regular basis. The alkalinity determined for Elendt M7 medium was 69.7 mg/L as NaHCO3, the hardness was 200 mg/L as CaCO3.
- Food during culturing: mixture of two species Pseudokirchneriella subcapitata : Desmodesmus subspicatus (in 2:1 ratio) originating from separate cultures in the Laboratory of Aquatic Toxicology. Group B vitamins and micronutrients necessary for proper growth were supplied with the lyophilized suspension of Spirulina sp.
- Feeding during test: no
- Health: Only organisms up to 24 hours old (not first brood progeny) and in good physiological condition were used in the tests.

ACCLIMATION
- Acclimation period: None; Elendt M7 medium was used to culture the test organism and as a diluent/solvent of the test item, and environmental conditions were the same as in the culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

200 mg/L as CaCO3

Test temperature:

19.1 - 21.1 ºC

pH:

- Start: 7.39 - 7.50
- End: 7.49 - 7.55

Dissolved oxygen:

- Start: 9.1 - 9.2 mg/L
- End: 8.7 - 8.9 mg/L

Conductivity:

518 µS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 0.63, 1.25, 2.5, 5.0 and 10 mg/L (based on range-finding test)
- Measured concentrations (t = 0h): - Measured concentrations (t = 48h):

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers of 150 mL capacity, covered with transparent lids
- Fill volume: 100 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Test medium: same as culture medium, see 'Details on test organisms'
- Intervals of water quality measurement: The pH values and the dissolved oxygen concentrations were measured in each treatment at exposure initiation before splitting up into replicates. At exposure termination, the pH values and the dissolved oxygen concentrations were measured in pooled replicates of each treatment

OTHER TEST CONDITIONS
- Photoperiod: daily cycle 16 h light : 8 h dark (KANLUX electronic time programmer, Poland)
- Light intensity: fluorescent lighting was used

EFFECT PARAMETERS MEASURED:
- Immobilization: Daphnia magna were observed for immobilization after 24 and 48 h of exposure. The Daphnia magna should be considered immobile if they showed no ability to swim within 15 seconds after swirling the test vessel.
- Other parameters: pH and the dissolved oxygen concentrations were measured in each treatment at exposure initiation before splitting up into replicates. At exposure termination, the pH values and the dissolved oxygen concentrations were measured in pooled replicates of each treatment.

RANGE-FINDING STUDY
- Test concentrations: 0, 0.0001, 0.001, 0.01, 0.1, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: No immobilization up to 1.0 mg/L, 100% immobilization at 10 mg/L after 48h exposure

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% C.I.: 2.99 - 3.78 mg/L

Details on results:

IMMOBILIZATION
In total, mean values for immobility after 48 h were 5%, 100% and 100% at concentrations of 2.5, 5.0 and 10 mg/L, respectively. Immobility was observed only in these three highest test concentrations. See 'Any other information on results incl. tables'.

Results with reference substance (positive control):

The 24-h EC50 for the reference substance was determined to be 1.01 mg/L; the 48-h EC50 was determined to be 0.92 mg/L.

Reported statistics and error estimates:

The endpoint values were calculated with the probit method. The lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) were estimated on the basis of statistical analyses (Step-down Cochran-Armitage Test Procedure). To make calculations and to conduct statistical analyses, the ToxRat Professional commercial software was used.

Table: Immobilization after 24 and 48 hours of exposure

Nominal test item concentration (mg/L)	Number of Daphnia magna	Number of immobilized Daphnia magna								Total of immobilized Daphnia magna [%]
		24 h				48 h
		Replicates
		A	B	C	D	A	B	C	D	24 h	48 h
Control	20	0	0	0	0	0	0	0	0	0	0
0.63	20	0	0	0	0	0	0	0	0	0	0
1.25	20	0	0	0	0	0	0	0	0	0	0
2.5	20	0	0	0	1	0	0	0	1	5	5
5.0	20	1	0	2	2	5	5	5	5	25	100
10	20	2	3	3	2	5	5	5	5	50	100

ADDITIONAL DETERMINED EFFECT VALUES

- 48-h NOEC: 2.5 mg/L

- 48-h LOEC: 5.0 mg/L

- 48-h EC20: 2.72 mg/L (95% C.I.: 2.34 - 3.05 mg/L)

- 48-h EC10: 2.43 mg/L (95% C.I. 2.04 - 2.75 mg/L)

Validity criteria fulfilled:

yes

Remarks:

see 'Additional information on materials and methods incl. tables'

Description of key information

The 48-h EC50 value is 3.36 mg/L in aquatic invertebrates (D. magna).

Additional information

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 0.63, 1.25, 2.5, 5.0 and 10 mg/L for 48 hours under static conditions. Test concentrations were based on a range-finding study. Analytical confirmation of nominal test concentrations showed that all test concentrations remained well within ±20% of nominal concentrations throughout the test. Therefore, effect concentrations are expressed as nominal. Immobilization was recorded after 24 and 48 hours exposure. No immobilization was observed in the 0.63 and 1.25 mg/L groups. At a test concentration of 2.5 mg/L cumulative mortality was 5%. Finally, in the 5.0 and 10 mg/L test groups 48-h after exposure 100% immobilization was observed. Based on these findings the 48-h EC50 value was determined at 3.36 mg/L.