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Short-term toxicity to fish

The experimental data from Haskell Laboratory for Toxicology and Industrial Medicinee following the standard testing procedures Haskell Laboratory for Toxicology and Industrial Medicine suggests that the 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride exhibits acute toxicity to fathead minnows under static unaerated test conditions during a 96-hour exposure. Its 96-hour LC-50  is 0.082 mg/l with 95% confidence limits of 0.065 mg/l and 0.11 mg/l. The slope of the dose response curve is 4.55 with 95% confidence limits of 2.16 and 6.94 is toxic to the aquatic environment.

Short-term toxicity to aquatic invertebrates

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.

Toxicity to aquatic algae and cyanobacteria

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.

Toxicity to micro-organism

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the closest read across substances, the toxicity on micro-organism predicted for 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride (Cas no. 548-62-9). Effect concentration i.e EC50 value estimated to be 4.577 mg/l for Tetrahymena pyriformis for 48 hrs duration. It can be concluded that the 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride (CAS: 548-62-9) likely to be toxic to micro-organism, hence it can be considered to benot classifiedas per the CLP classification criteria for aquatic environment.

Additional information

Short-term toxicity to fish

Following four studies of target substance includes experimental data following standard procedure, data from peer reviewed journal, predicted data from Authoritative database are summarized below to conclude the toxicity of N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride towards fish:

The experimental data from Haskell Laboratory for Toxicology and Industrial Medicinee following the standard testing procedures Haskell Laboratory for Toxicology and Industrial Medicine suggests that the 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride exhibits acute toxicity to fathead minnows under static unaerated test conditions during a 96-hour exposure. Its 96-hour LC-50  is 0.082 mg/l with 95% confidence limits of 0.065 mg/l and 0.11 mg/l. The slope of the dose response curve is 4.55 with 95% confidence limits of 2.16 and 6.94 is toxic to the aquatic environment.

The above study is supported by the result available from peer reviewed journal The Journal of Toxicological Sciences 1982, indicates that

The Lethal concentration to 50% of Oryzias latipes for 24 Hour under static condition is 0.2 mg/L and for 48 hr is 0.1 mg/L. It can be concluded that the [4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride is toxic to the aquatic environment.

Another supporting study by the predicted data from Danish QSAR database estimates, The lethal concentration (LC50) at 96 hrs for [4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride in Fathead minnow Using three different models i.e, Battery, Leadscope and SciQSAR is estimated to be 0.104 mg/L.

It is further supported by the predicted data by ACD (Advanced Chemistry Development)/I-Lab predictive module  (v5.0.0.184) the predicted Median Lethal Concentration (LC50, mg/L) for fish species Pimephales promelas of the [4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride found to be 0.13 mg/l on the basis of mortality effects.

Thus based on the effect concentrations which is in the range 0.082 mg/L to 0.2 mg/l give the conclusion that test substance N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (Cas no. 548-62-9) was likely to be toxic to fish at environmentally relevant concentrations and can be considered to beAquatic Acute 1as per the CLP classification criteria.

Short-term toxicity to aquatic invertebrates

Following study of target substance includes experimental data following standard procedure is summarized 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.

Toxicity to aquatic algae and cyanobacteria

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 ErC50. 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 EyC50.

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 ErC50(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 EyC50(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 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.

Toxicity to micro-organism

Following studies for target substance includes predicted data fromvalidated toolbox and experimental data of read across are summarized below to conclude the toxicity of 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride (CAS: 548-62-9) towards micro-organism:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the closest read across substances, the toxicity on aquatic algae predicted for 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride (Cas no. 548-62-9). Effect concentration i.e EC50 value estimated to be 4.577 mg/l for Tetrahymena pyriformis for 48 hrs duration. It can be concluded that the 4-{bis[4-(dimethylamino)phenyl{methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride (CAS: 548-62-9) likely to be toxic to mico-organism, hence it can be considered to benot classifiedas per the CLP classification criteria for aquatic environment.

The availabe experimental study for read across Menadione (CAS: 58 -27 -5) from Saudi Journal of Biological Sciences 2017, suggests that

The minimal inhibitory concentration (MIC) of Staphylococcus aureus for Menadione in 24 h is 0.128 mg/L. It ca be concluded that the Menadione is toxic to the aquatic microorganism.

Thus based on the effect concentrations which is in the range 0.128 mg/L mg/l to 4.577 give the conclusion that test substance 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride (CAS: 548-62-9) was likely to be toxic to micro-organism at environmentally relevant concentrations and can be considered to beAquatic Acute 1as per the CLP classification criteria.