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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

25 October 2010 - 02 November 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Data is from experimental reports following standard testing procedures.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Section 2, No. 201, "Fresh water Alga and Cyanobacteria, Growth Inhibition Test", adopted on 23 March 2006.

Principles of method if other than guideline:

Freshwater Alga, Growth Inhibition Test of 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride in fresh water Unicellular green alga, Pseudokirchneriella subcapitata (Selenastrum capricornutum) was performed in compliance with the OECD Guidelines for Testing of Chemicals, Section 2, No. 201.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name (IUPAC): 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride
- Name of test material (as cited in study report): Gentian Violet
- Molecular formula: C25H30ClN3
- Molecular weight: 407.986 g/mol
- Smiles notation): CN(C)C1=CC=C(C=C1)C(=C2C=CC(=[N+](C)C)C=C2)C3=CC=C(C=C3)N(C)C.[Cl-]
- InChl : InChI=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

Analytical monitoring:

not required

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Strain: ATCC-22662
Source: American Type Culture Collection (ATCC) 10801 university Boulevard Manassas, Virginia 20110-2209.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

Culture maintained at 25.8 to 26.00C.

pH:

7.63 to 8.13

Nominal and measured concentrations:

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

Details on test conditions:

Duration : 72 hours.
Culture Medium : OECD Medium
Initial Cell Concentration : 25000 cell/ml
Test Flasks : The cultures were maintained and tested in 250 ml conical flasks each containing 100 ml OECD Medium
Culture Apparatus : Culture maintained at 25.8 to 26.00C.
Illumination : A continuous uniform illumination of 5608 to 5668 Lux, cool white light during the test
In positive control group percent inhibition of growth in fresh water unicellular green alga, Pseudokirchneriella subcapitata at 72 h, was 21.01%, 65.18% and 90.97% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence level: 0.11 to 0.40 mg/L

Details on results:

Exposure Concentration
Exponentially growing cultures of the algae were exposed to test article at the concentrations levels of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L for a period of 72 hours, along with a concurrent untreated control culture of the algae.Potassium Dichromate, serving as a positive control, was also concurrently tested at concentrations levels of 0.4 mg, 0.8 mg and 1.6 mg/L.
 
Cell Concentration
For initial cell concentration, cell count was taken from the mother culture then exact 5681 l of culture was added to medium to make initial cell concentration 25000 cells/ml in test container. The average algal cell counts of the test solutions and the values of percentage growth inhibition for the test and the positive control are presented in Table 1. The data on cell concentration for each test flask at each measuring point for the test and positive controls are presented in Appendix A.
 
Growth Inhibition
In the present study, percent inhibition of growth in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 3.62%, 5.82%, 9.82%, 20.88%, 47.95 and 72.83 % recorded at the concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 and 0.8 mg/L respectively. In positive control grouppercent inhibition of growth in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 21.01%, 65.18% and 90.97% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively (Table 2).

Yield
In the present study, percent inhibition in yield in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 10.45%, 16.37%, 25.64%, 47.73%, 79.29% and 92.90% recorded at the concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L respectively. In positive control grouppercent inhibition of the yield in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 48.13%, 89.74% and 98.22% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively (Table 2).
 
Validity of the Test
The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures must not exceed 7% in test withPseudokirchneriella subcapitata.In present study the coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures was 0.010%.
 
Evaluation of ErC50 and EyC50 Value
On the basis of observations made during this test, the ErC50(72h) ofC. I. Basic Violet 3inducing inhibition of the growth of fresh water unicellular green algaePseudokirchneriella subcapitatawas found to be 0.42 mg/L with fiducial limits between 0.19 to 0.93 mg/L at 95% confidence level. On the basis of observations made during this test, the EyC50(72h) ofC. I. Basic Violet 3inducing inhibition of the yield of fresh water unicellular green algaePseudokirchneriella subcapitatawas found to be 0.21 mg/L with fiducial limits between 0.11 to 0.40 mg/L at 95% confidence level. On the basis of observations made during this test, the ErC50(72h) of positive control,PotassiumDichromate,inducing inhibition of the growth of freshwater unicellular green algaePseudokirchneriella subcapitatawas found to be 0.66 mg/L with fiducial limits between  0.33 to 1.32 mg/L at 95% confidence level. On the basis of observations made during this test, the EyC50(72h) of positive control,PotassiumDichromate,inducing inhibition of the yield of freshwater unicellular green algaePseudokirchneriella subcapitatawas found to be 0.41 mg/L with fiducial limits between 0.16 to 1.06 mg/L at 95% confidence level.

TABLE 1

AVERAGE GROWTH RATE

Group	Test Concentration (mg/L)		Average Growth Rate At
			24 Hour	48 Hour	72 Hour	0-72 Hours
Blank Control	0	Mean	1.3167	1.1419	0.4258	0.9615
		SD	0.04	0.09	0.08	0.010
		CV	0.03	0.08	0.19	0.010
		%CV	3.09	8.01	19.26	1.018
Test Article (C. I. Basic Violet 3)	0.025	Mean	1.1728	1.0465	0.5609	0.9267
		SD	0.05	0.14	0.06	0.015
		CV	0.04	0.13	0.11	0.017
		%CV	4.00	13.20	11.15	1.668
	0.05	Mean	1.2428	0.8774	0.5964	0.9055
		SD	0.03	0.12	0.13	0.009
		CV	0.03	0.14	0.21	0.010
		%CV	2.66	13.78	21.13	1.007
	0.1	Mean	1.0805	0.9552	0.5657	0.8671
		SD	0.15	0.20	0.16	0.045
		CV	0.13	0.21	0.29	0.052
		%CV	13.48	21.35	28.76	5.217

Group	Test Concentration (mg/L)		Average Growth Rate At
			24 Hour	48 Hour	72 Hour	0-72 Hours
Test Article (C. I. Basic  Violet 3)	0.2	Mean	0.8891	0.5580	0.8351	0.7607
		SD	0.02	0.13	0.04	0.035
		CV	0.03	0.23	0.04	0.045
		%CV	2.65	23.04	4.45	4.550
	0.4	Mean	0.9158	0.3819	0.2037	0.5005
		SD	0.04	0.10	0.16	0.030
		CV	0.04	0.26	0.77	0.059
		%CV	4.37	25.82	77.49	5.938
	0.8	Mean	0.1510	0.5327	0.1001	0.2613
		SD	0.10	0.23	0.26	0.039
		CV	0.64	0.43	2.59	0.150
		%CV	63.88	43.18	259.38	14.967
Positive control (Potassium Dichromate)	0.4	Mean	0.7249	0.9205	0.6332	0.7595
		SD	0.06	0.04	0.13	0.011
		CV	0.08	0.05	0.21	0.015
		%CV	7.59	4.60	20.57	1.457
	0.8	Mean	0.1987	0.4012	0.4046	0.3348
		SD	0.18	0.08	0.17	0.018
		CV	0.90	0.20	0.43	0.055
		%CV	90.12	20.27	43.17	5.521
	1.6	Mean	-0.1703	0.3750	0.0557	0.0868
		SD	0.29	0.30	0.05	0.026
		CV	-1.73	0.80	0.87	0.296
		%CV	-173.21	80.21	86.83	29.608

SD – Standard Deviation, CV - Coefficient of variation,

 %CV – Percentage of Coefficient of variation

TABLE 2 

PERCENTAGE GROWTH INHIBITION & INHIBITION IN YIELD OF ALGA

Group	Test Concentration (mg/L)	Growth Inhibition (%)	Inhibition Yield (%)
BlankControl	0	0	0
Test Article (C. I. Basic Violet 3)	0.025	3.62	10.45
	0.05	5.82	16.37
	0.1	9.82	25.64
	0.2	20.88	47.73
	0.4	47.95	79.29
	0.8	72.83	92.90
Positive control (Potassium Dichromate)	0.4	21.01	48.13
	0.8	65.18	89.74
	1.6	90.97	98.22

Validity criteria fulfilled:

yes

Conclusions:

On the basis of observations made during this test, the ErC50 (72h) of C. I. Basic Violet 3 inducing inhibition of the growth of fresh water unicellular green algae Pseudokirchneriella subcapitata was found to be 0.42 mg/L with fiducial limits between 0.19 to 0.93 mg/L at 95% confidence level.
On the basis of observations made during this test, the EyC50 (72h) of C. I. Basic Violet 3 inducing inhibition of the yield of fresh water unicellular green algae Pseudokirchneriella subcapitata was found to be 0.21 mg/L with fiducial limits between 0.11 to 0.40 mg/L at 95% confidence level.
It can be concluded from EcC50 and EyC50 value that the 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride is toxic to the aquatic environment.

Executive summary:

Freshwater Alga, Growth Inhibition Test of 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride in fresh water Unicellular green alga,Pseudokirchneriella subcapitata(Selenastrum capricornutum) was performed in compliance with the OECD Guidelines for Testing of Chemicals, Section 2, No. 201, "Fresh water Alga and Cyanobacteria, Growth Inhibition Test".

4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride was tested for its possible inhibitory effect on growth of alga, Pseudokirchneriella subcapitata. Exponentially growing cultures of the algae were exposed to test article at concentrations levels of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 and 0.8 mg/L for a period of 72 hours, along with a concurrent blank control. Potassium Dichromate, serving as a positive control, was also concurrently tested at concentrations levels of 0.4 mg, 0.8 mg and 1.6 mg/L. The cell concentration, i.e. the number of cells per ml, for all the concentration levels, was counted at 24, 48 and 72 hours of start of the exposure by counting them under microscope in a haemocytometer counting chamber and the inhibition of growth in relation to the control culture was determined. 

The endpoint was inhibition of growth, expressed as the logarithmic increase in biomass (average specific growth rate) during the exposure period. From the average specific growth rates recorded in a series of test solutions, the concentration bringing about 50% inhibition of growth rate was determined and expressed as the E_rC₅₀. The additional endpoint was inhibition of yield, expressed as the biomass at the end of exposure period minus the biomass at the start of the exposure period. From the yield recorded in a series of test solutions, the concentration bringing about 50% inhibition of yield was determined and expressed as the E_yC₅₀.

In the present study, percent inhibition of growth in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 3.62%, 5.82%, 9.82%, 20.88%, 47.95 and 72.83 % recorded at the concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 and 0.8 mg/L respectively. In positive control grouppercent inhibition of growth in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 21.01%, 65.18% and 90.97% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively.

 

In the present study, percent inhibition in yield in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 10.45%, 16.37%, 25.64%, 47.73%, 79.29% and 92.90% recorded at the concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L respectively. In positive control grouppercent yield in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 48.13%, 89.74% and 98.22% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively. On the basis of observations made during this test, the E_rC₅₀(72h) of 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride inducing inhibition of the growth of fresh water unicellular green algaePseudokirchneriella subcapitatawas found to be 0.42 mg/L with fiducial limits between 0.19 to 0.93 mg/L at 95% confidence level. On the basis of observations made during this test, the E_yC₅₀(72h) of 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloridei nducing inhibition of the yield of fresh water unicellular green algaePseudokirchneriella subcapitatawas found to be 0.21 mg/L with fiducial limits between 0.11 to 0.40 mg/L at 95% confidence level.

It can be concluded from EcC50 and EyC50  value that the 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride is toxic to the aquatic environment and can be considered as "Aquatic Acute 1" as per CLP regulation.

Description of key information

On the basis of observations made during this test, the ErC50 (72h) of C. I. Basic Violet 3 inducing inhibition of the growth of fresh water unicellular green algae Pseudokirchneriella subcapitata was found to be 0.42 mg/L with fiducial limits between 0.19 to 0.93 mg/L at 95% confidence level. On the basis of observations made during this test, the EyC50 (72h) of C. I. Basic Violet 3 inducing inhibition of the yield of fresh water unicellular green algae Pseudokirchneriella subcapitata was found to be 0.21 mg/L with fiducial limits between 0.11 to 0.40 mg/L at 95% confidence level. It can be concluded from EcC50 and EyC50  value that the 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride is toxic to the aquatic environment.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.21 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 algae and cyanobacteria:

Freshwater Alga, Growth Inhibition Test of4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloridein fresh water Unicellular green alga,Pseudokirchneriella subcapitata(Selenastrum capricornutum)was performed in compliance with the OECD Guidelines for Testing of Chemicals, Section 2, No. 201, "Fresh water Alga and Cyanobacteria, Growth Inhibition Test".

4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloridewas tested for its possible inhibitory effect on growth of alga, Pseudokirchneriella subcapitata. Exponentially growing cultures of the algae were exposed to test article at concentrations levels of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 and 0.8 mg/L for a period of 72 hours, along with a concurrent blank control.Potassium Dichromate, serving as a positive control, was also concurrently tested at concentrations levels of 0.4 mg, 0.8 mg and 1.6 mg/L. The cell concentration, i.e. the number of cells per ml, for all the concentration levels, was counted at 24, 48 and 72 hours of start of the exposure by counting them under microscope in a haemocytometer counting chamber and the inhibition of growth in relation to the control culture was determined. 

The endpoint was inhibition of growth, expressed as the logarithmic increase in biomass (average specific growth rate) during the exposure period. From the average specific growth rates recorded in a series of test solutions, the concentration bringing about 50% inhibition of growth rate was determined and expressed as the E_rC₅₀. The additional endpoint was inhibition of yield, expressed as the biomass at the end of exposure period minus the biomass at the start of the exposure period. From the yield recorded in a series of test solutions, the concentration bringing about 50% inhibition of yield was determined and expressed as the E_yC₅₀.

In the present study, percent inhibition of growth in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 3.62%, 5.82%, 9.82%, 20.88%, 47.95 and 72.83 % recorded at the concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 and 0.8 mg/L respectively. In positive control grouppercent inhibition of growth in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 21.01%, 65.18% and 90.97% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively.

 

In the present study, percent inhibition in yield in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 10.45%, 16.37%, 25.64%, 47.73%, 79.29% and 92.90% recorded at the concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L respectively. In positive control grouppercent yield in fresh water unicellular green alga,Pseudokirchneriella subcapitataat 72 h, was 48.13%, 89.74% and 98.22% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively.On the basis of observations made during this test, the E_rC₅₀(72h) of4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chlorideinducing inhibition of the growth of fresh water unicellular green algaePseudokirchneriella subcapitatawas found to be 0.42 mg/L with fiducial limits between 0.19 to 0.93 mg/L at 95% confidence level.On the basis of observations made during this test, the E_yC₅₀(72h) of4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloridei nducing inhibition of the yield of fresh water unicellular green algae Pseudokirchneriella subcapitatawas found to be 0.21 mg/L with fiducial limits between 0.11 to 0.40 mg/L at 95% confidence level.

It can be concluded from EcC50 and EyC50  value that the 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride is toxic to the aquatic environment and can be considered as "Aquatic Acute 1" as per CLP regulation.