[4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride

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:

25 October 2010 - 31 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Data is from experimental reports following standard testing procedures.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Guidelines for Testing of Chemicals, Section 2, No. 202, "Daphnia Sp., Acute Immobilisation Test" adopted 13th April 2004

Principles of method if other than guideline:

Acute Immobilisation test in Daphnia magna was performed in compliance with the OECD Guidelines for Testing of Chemicals, Section 2, No. 202, "Daphnia Sp., Acute Immobilisation Test"

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Crystal violet
- IUPAC name: N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride
- Molecular formula : C25H30N3.Cl
Molecular weight : 407.986 g/mol
- Smiles notation: C(\c1ccc(N(C)C)cc1)(c1ccc(N(C)C)cc1)=C1\C=C\C(=[N+](/C)C)C=C1.[ClH-]
- InChl:1S/C25H30N3.ClH/c1-26(2)22-13-7-19(8-14-22)25(20-9-15-23(16-10-20)27(3)4)21-11-17-24(18-12-21)28(5)6;/h7-18H,1-6H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid
- Purity: > 80%

Test organisms (species):

Daphnia magna

Details on test organisms:

Age at beginning of the test: Less than 24 hours
No. of Daphnids per Concentration: 20 (5 Daphnids per replicate)
Light : 16 hour light 8 hour dark
Feeding: No feeding during the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

246.96 mg/L (as CaCO3)

Test temperature:

18.5 to 19.5 0C

pH:

8.41 to 8.81

Dissolved oxygen:

5.35 to 5.96 mg/L

Nominal and measured concentrations:

0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L.

Details on test conditions:

Light: 16 hour light 8 hour dark
Test containers:100 ml beakers, each containing five Daphnids
Test water: Reconstituted water
Loading: 10 ml of test solution / animal
Culture/Incubation Apparatus: Culture maintained at 18 to 220C

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: conf. level 0.24 to 0.50 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: conf. level 0.41 to 0.69 mg/L

Results with reference substance (positive control):

POSITIVE CONTROL : Potassium Dichromate
The acute immobilisation EC50 value of positive control, Potassium Dichromate, to Daphnia magna at 24 hour exposure period was found to be 0.63 mg/L with fiducial limits between 0.52 to 0.77 mg/L at 95% confidence level.
The acute immobilisation EC50 value of positive control, Potassium Dichromate, to Daphnia magna at 48 hour exposure period was found to be 0.53 mg/L with fiducial limits between 0.44 to 0.64 mg/L at 95% confidence level.

TABLE 1

IMMOBILISATION IN DAPHNIA EXPOSED TOC. I. BASIC VIOLET 3

Dose Group	Concentration (mg/L)	No. of Daphnids	24 hours		48 hours (Cumulative)
			Nos. Immobilized	% Immobilized	Nos. Immobilized	% Immobilized
Blank Control	0	20	0	0	0	0
Test article (C. I. Basic Violet 3)	0.025	20	0	0	0	0
	0.05	20	0	0	0	0
	0.1	20	0	0	2	10
	0.2	20	2	10	5	25
	0.4	20	5	25	11	55
	0.8	20	15	75	17	85
Positive control (Potassium Dichromate)	0.4	20	4	20	5	25
	0.63	20	10	50	13	65
	1	20	16	80	20	100

TABLE 2

PHYSICOCHEMICAL CHARACTERISTICS OF THE Test SOLUTION

Dose Group	Concentration (µg/L)	Parameters	0 h	48 h
				R1	R2	R3	R4
Blank Control	0	pH	8.41	8.52	8.48	8.55	8.61
		D. O. (mg/L)	5.96	5.88	5.81	5.78	5.75
		Temp.^0C	19.5	18.5	18.5	18.5	18.5
Test article (C. I. Basic Violet 3)	0.8	pH	8.41	8.78	8.81	8.65	8.72
		D. O. (mg/L)	5.96	5.74	5.80	5.65	5.55
		Temp.^0C	19.5	18.5	18.5	18.5	18.5
Positive control (Potassium Dichromate)	1	pH	8.41	8.64	8.55	8.76	8.61
		D. O. (mg/L)	5.96	5.45	5.61	5.35	5.45
		Temp.^0C	19.5	18.5	18.5	18.5	18.5

TABLE 3

RESULTS OF CONCENTRATION VERIFICATION(C. I. Basic Violet 3)

Study Day (Hrs)	Nominal Concentration of test article (mg/L)	Replicate No.	Actual Concentration of test article determined (mg/L)	% Error in Concentration
1 (0 Hrs)	0.025	1	0.024	-4.39
	0.025	2	0.024	-4.65
	0.025	3	0.024	-4.67
	Average	-	0.024	-4.57
	SD	-	0.00	0.16
	% RSD	-	0.17	-3.47
	0.8	1	0.73	-8.41
	0.8	2	0.74	-7.87
	0.8	3	0.74	-8.02
	Average	-	0.74	-8.10
	SD	-	0.00	0.28
	% RSD	-	0.30	-3.46

Study Day (Hrs)	Nominal Concentration of test article (mg/L)	Replicate No.	Actual Concentration of test article determined (mg/L)	% Error in Concentration
3 (48 Hrs)	0.025	1	0.017	-29.61
	0.025	2	0.017	-28.94
	0.025	3	0.017	-29.82
	Average	-	0.017	-29.46
	SD	-	0.00	0.46
	% RSD	-	0.065	-1.55
	0.8	1	0.46	-36.90
	0.8	2	0.47	-36.21
	0.8	3	0.47	-36.70
	Average	-	0.47	-36.60
	SD	-	0.00	0.36
	% RSD	-	0.56	-0.98

 

Validity criteria fulfilled:

yes

Conclusions:

The acute immobilisation EC50value of N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride to Daphnia magna at 24 hour exposure period was found to be 0.53 mg/L with fiducial limits between 0.41 to 0.69 mg/L at 95% confidence level and 4at 8 hour exposure period was found to be 0.35 mg/L with fiducial limits between 0.24 to 0.50 mg/L at 95% confidence level.
It can be concluded forom EC50 value obtained it can be concluded that the substance N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride is toxic to the aquatic environment.

Executive summary:

Acute Immobilisation test in Daphnia magna was performed in compliance with the OECD Guidelines for Testing of Chemicals, Section 2, No. 202, "Daphnia Sp., Acute Immobilisation Test" . N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride was tested for acute toxic effects on the swimming capabilities of Daphnia magna employing a static test procedure.

In the main study, six groups of daphnids, each comprising 5 daphnids per replicate, were exposed to the test article at concentration levels of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L. Observations were made for immobilisation at 24 hours and 48 hours of treatment.

A concurrent blank control was also run simultaneously. Potassium Dichromate was employed as a positive control and tested at the concentration of   0.4 mg, 0.63 mg and 1 mg/L.

The animals were observed for immobility after 24 hours and 48 hours of exposure. Those daphnids which were unable to swim within 15 seconds after gentle agitation of the test container were considered to be immobile.

The rates of immobilisation observed at 24 hours were 0%, 0%, 0%, 10%, 25% and 75% at the test concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L respectively.

The rates of immobilisation observed at 48 hours were 0%, 0%, 10%, 25%, 55% and 85% at the test concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L respectively.

The Positive control, Potassium Dichromate induced immobilisation in 20%, 50% and 80% of the exposed daphnids at 24 hours and 25%, 65% and 100% immobilization at 48 hours at the concentrations of 0.4 mg, 0.63 mg and 1 mg/L respectively. No immobilisation was observed in blank control group.

The acute immobilisation EC₅₀value of N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride to Daphnia magna at 24 hour exposure period was found to be 0.53 mg/L with fiducial limits between 0.41 to 0.69 mg/L at 95% confidence level.

The acute immobilisation EC₅₀value of N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride to Daphnia magna at 48 hour exposure period was found to be 0.35 mg/L with fiducial limits between 0.24 to 0.50 mg/L at 95% confidence level.

On the basis of EC50 value obtained it can be concluded that the substance N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride is toxic to the aquatic environment and can be considered as "Aquatic Acute 1" as per CLP regulation.

Description of key information

The acute immobilisation EC50 value of N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride to Daphnia magna at 24 hour exposure period was found to be 0.53 mg/L with fiducial limits between 0.41 to 0.69 mg/L at 95% confidence level and at 48 hour exposure period was found to be 0.35 mg/L with fiducial limits between 0.24 to 0.50 mg/L at 95% confidence level.

It can be concluded forom EC50 value obtained it can be concluded that the N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride is toxic to the aquatic environment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.35 mg/L

Additional information

Following study of target substance includes experimental data following standard procedure issummarized below to conclude the toxicity of

N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride towards aquatic invertebrate:

The acute immobilisation EC50 value of N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride to Daphnia magna at 24 hour exposure period was found to be 0.53 mg/L with fiducial limits between 0.41 to 0.69 mg/L at 95% confidence level and at 48 hour exposure period was found to be 0.35 mg/L with fiducial limits between 0.24 to 0.50 mg/L at 95% confidence level.

It can be concluded forom EC50 value obtained it can be concluded that the N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride is toxic to the aquatic environment.