2,4,6-trichloroaniline

Endpoint summary

Description of key information

Short term toxicity to fish:

Study was conducted to access the effect of test chemical 2,4,6-trichloroaniline on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). The nominal concentration selected for the experiment were 0.8 mg/L, 1.76 mg/L, 3.87mg/L, 8.5mg/L, 18.72mg/L, respectively and Zebra Fish Danio rerio were exposed to these concentration for 96 hours. The test substance was slightly soluble in water. Therefore, the stock solution was prepared by dissolving 1gm of the test substance in 1 liters of potable water (passed through reverse osmosis system) and kept it for 30min of sonication. After the completion of the sonication process, the sample was run through HPLC system for getting the actual water solubility of the test substance. The actual solubility obtained was 48.89 mg/L. From this stock solution further test concentrations were prepared for achieving test concentrations of 0.8 mg/L, 1.76 mg/L, 3.87mg/L, 8.5mg/L, 18.72mg/L, respectively. Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. The fishes were moving slowly as compared to control. No mortalities were observed in the control aquaria.  

LC0 (96 hours) (highest loading at which no mortality was observed) = 8.51mg/L

LC50 (96 hours) Experimental = >8.51mg/L & <18.72mg/L

LC100 (96 hours) (lowest loading at which 100% mortality was observed) = 18.72mg/L

LC50 (through probit analysis) = 10.06mg/L

After rounding-off the numerical values, the LC 50 was determine to be 10 mg/L. Based on the LC50 value (10 mg/l), chemical 2,4,6-trichloroaniline, consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates:

Acute juvenile immobilization and embronic hatching inhibition assay was performed to study the adverse effects of 2, 4, 6 trichloroaniline on daphnia. Test performed in accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). At least five test concentrations were used. N,N- Dimethylformamide (DMF) were used as a solvent. Before test daphnia acclimated and daphnia showed no signs of stress such as high mortality, presence of males and ephippia, delay in the production of first brood, discoloured animals, etc. Young female juveniles were exposed to the test solution and the immobilized juveniles were observed after 24 and 48 hrs and the embronic hatching inhibition after 72 hrs. After the exposure of chemical with daphnia magna effect were observed on the juvenile immobilization and embronic hatching inhibition. The EC50 was determine to be 2.3 mg/l based on the immobilisation and 0.83 on the basis of embronic hatching inhibition for 3 days. Based on the EC50 (2.3 mg/l) chemical concluded to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

study was designed to assess the toxic effects of the test compound 2,4,6-trichloroaniline on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance 2,4,6-trichloroaniline was prepared by adding 250mg of test substance in 250ml of BBM and this stock solution was sonicated for 30 minutes. The observed concentration was checked for this chemical stock solution and the resulting final concentration of the stock solution was found out to be 67.5mg/l. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. After 72 hours of exposure to 2,4,6-trichloroaniline to various nominal test concentration, EC50 was determine to be at 4.445mg/l, through equation and graphically respectively through probit analysis. Based on the above EC50 value, chemical was consider as toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

Toxicity to microorganisms:

study was designed to access the effect of the substance 2,4,6-trichloroaniline on the luminescent bacteria Photobacterium phosphoreum carried out for 30 minutes. Before testing chemical was analyzed by HPLC. Based on the inhibition of luminescence of Photobacterium phosphoreum, the EC50 was observed at 4.61 mg/l. Based on the EC50, chemical can be consider as toxic.

 

Additional information

Summarized result of toxicity of the chemical2,4,6-trichloroaniline (CAS no. 634-93-5) on the growth of fishes, aquatic invertebrates and algae and microorganisms was studied by considering and collecting the data from various databases for target chemical. The studies are as follows:  

 

Short term toxicity to fish:

Based on the experimental data from various database for the target chemical study have been reviewed to determine the mode of action of2,4,6-trichloroaniline (CAS no. 634-93-5) on the mortality rate and behavior of fish. The studies are as mentioned below:

 

In the first key study from experimental report 2015, study was conducted to access the effect of test chemical 2,4,6-trichloroaniline on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). The nominal concentration selected for the experiment were 0.8 mg/L, 1.76 mg/L, 3.87mg/L, 8.5mg/L, 18.72mg/L, respectively and Zebra Fish Danio rerio were exposed to these concentration for 96 hours. The test substance was slightly soluble in water. Therefore, the stock solution was prepared by dissolving 1gm of the test substance in 1 liters of potable water (passed through reverse osmosis system) and kept it for 30min of sonication. After the completion of the sonication process, the sample was run through HPLC system for getting the actual water solubility of the test substance. The actual solubility obtained was 48.89 mg/L. From this stock solution further test concentrations were prepared for achieving test concentrations of 0.8 mg/L, 1.76 mg/L, 3.87mg/L, 8.5mg/L, 18.72mg/L, respectively. Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. The fishes were moving slowly as compared to control. No mortalities were observed in the control aquaria.  

LC0 (96 hours) (highest loading at which no mortality was observed) = 8.51mg/L

LC50 (96 hours) Experimental = >8.51mg/L & <18.72mg/L

LC100 (96 hours) (lowest loading at which 100% mortality was observed) = 18.72mg/L

LC50 (through probit analysis) = 10.06mg/L

After rounding-off the numerical values, the LC 50 was determine to be 10 mg/L. Based on the LC50 value (10 mg/l), chemical 2,4,6-trichloroaniline, consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

First key study was supported by the second study from authoritative database 2018. Study was conducted to determine the effect of test chemical on the mortality of fish for 96 hrs. Test performed according to the OECD Guideline 203 (Fish, Acute Toxicity Test). The LC50 (96 h) value of the test substance 2,4,6-trichloroaniline to fish is determine to be 5.3 mg/L. Thus considering the CLP Criteria for aquatic classification of the substance, it is concluded that 2,4,6-trichloroaniline exhibits short term toxicity to fish and is therefore classified as Aquatic Chronic category 2.

 

Similarly short term toxicity of test chemical was studied on the mortality rate of fish. Three gallon glass aquaria each holding 10 L of water were used. 10, 1, 0.1 mg/L concentrations prepared. 2 vessels for each concentration were used. The LC50 value of the test substance 2,4,6-trichloroaniline to Pimephales promelas is determine to be 1-10 mg/L on the basis of mortality. Thus considering the CLP Criteria for aquatic classification of the substance, it is concluded that 2,4,6-trichloroaniline exhibits short term toxicity to fish and is therefore classified as Aquatic Chronic category 2.

 

In the fourth supporting study from secondary sources 2018, short term toxicity of test chemical was studied on the mortality rate of fish. After the exposure of chemical with fish mortality were observed. On the basis of mortality observed, the LC50 value for the test substance 2,4,6-trichloroaniline to Leuciscus idus is observed to be 2.3 mg/L. Based on the LC50, chemical was consider as toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

Based on the overall studies from experimental report 2015, authoritative databases and secondary sources 2018, it was concluded that the chemical 2,4,6-trichloroaniline was toxic and consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

 

Short term toxicity to aquatic invertebrates:

Based on the experimental data from various database for the target chemical study have been reviewed to determine the mode of action of2,4,6-trichloroaniline (CAS no. 634-93-5)on the mobility rate and behavior of aquatic invertebrates. The studies are as mentioned below:

 

In the first key study from peer reviewed journal 2001, Acute juvenile immobilization and embronic hatching inhibition assay was performed to study the adverse effects of 2, 4, 6 trichloroaniline on daphnia. Test performed in accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). At least five test concentrations were used. N,N- Dimethylformamide (DMF) were used as a solvent. Before test daphnia acclimated and daphnia showed no signs of stress such as high mortality, presence of males and ephippia, delay in the production of first brood, discoloured animals, etc. Young female juveniles were exposed to the test solution and the immobilized juveniles were observed after 24 and 48 hrs and the embronic hatching inhibition after 72 hrs. After the exposure of chemical with daphnia magna effect were observed on the juvenile immobilization and embronic hatching inhibition. The EC50 was determine to be 2.3 mg/l based on the immobilisation and 0.83 on the basis of embronic hatching inhibition for 3 days. Based on the EC50 (2.3 mg/l) chemical concluded to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

First key study was supported by the second study from authoritative database 2018. Study was conducted to determine the effect of test chemical on the growth of aquatic invertebrates for 48 hrs. Test performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Based on the immobility of daphnia magna due to the chemical effect, the EC50 was determine to be 4.3 mg/l. Thus considering the CLP Criteria for aquatic classification of the substance, it is concluded that 2,4,6-trichloroaniline exhibits short term toxicity to aquatic invertebrate and is therefore classified as Aquatic Chronic category 2.

 

Similarly above results and classification was supported by another studies from secondary sources 2018. Study was conducted to determine the effect of test chemical on the growth of aquatic invertebrates. After the exposure of test chemical effect were observed and measured at the time when 50 % population affected. Based on the Distance moved, change in direct movement, the EC50 was determine to be 6 mg/l.

 

Based on the overall studies from peer reviewed journal, authoritative databases and secondary sources 2018, it was concluded that the chemical 2,4,6-trichloroaniline was toxic to the aquatic invertebrates and consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

 

Toxicity to aquatic algae and cyanobacteria:

Based on the experimental data from various database for the target chemical study have been reviewed to determine the mode of action of2,4,6-trichloroaniline (CAS no. 634-93-5)on the growth of aquatic algae and cyanobacteria. The studies are as mentioned below:

 

In the first key study from experimental report 2015, study was designed to assess the toxic effects of the test compound 2,4,6-trichloroaniline on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance 2,4,6-trichloroaniline was prepared by adding 250mg of test substance in 250ml of BBM and this stock solution was sonicated for 30 minutes. The observed concentration was checked for this chemical stock solution and the resulting final concentration of the stock solution was found out to be 67.5mg/l. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. After 72 hours of exposure to 2,4,6-trichloroaniline to various nominal test concentration, EC50 was determine to be at 4.445mg/l, through equation and graphically respectively through probit analysis. Based on the above EC50 value, chemical was consider as toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

First key study was supported by the second study from peer reviewed journal 2007, Aim of this study was access the effect of test chemical on the growth of aquatic algae and cyanobacteria. The toxic effects of substituted anilines on Pseudokirchneriella subcapitata with the use of a closed algal toxicity testing technique with no headspace was performed. Two response endpoints (i.e., dissolved oxygen production [DO] and algal growth rate) were used to evaluate the toxicity of anilines. Both DO and growth rate endpoints revealed similar sensitivity to the effects of anilines. However, 2, 4, 6 trichloroaniline showed stronger inhibitory effects on microalgal photosynthetic reactions than that on algal growth. EC50 values for the microalgae Pseudokirchneriella subcapitata were determine to be equal to 4.07 mg/L (ΔDO) and 6.32 mg/L (growth rate) respectively. For various aquatic organisms, the relative sensitivity relationship for the test compound is Daphnia magna> luminescent bacteria (Microtox) >Pocelia reticulata>Pseudokirchneriella subcapitata> fathead minnow >Tetrahymena pyriformis. QSAR values calculated on the basis of Elumo, log KOW, or both values is determine to be 1.68 mg/L (ΔDO) and 1.49 mg/L (growth rate) respectively.

 

Similarly another study was designed (from peer reviewed journal 2007) to access the effect of the substance 2,4,6-trichloroaniline on the growth of green alga Pseudokirchneriella subcapitata carried out for 96 hrs. The test organism used for the study is Pseudokirchneriella subcapitata, UTEX 1648. Before commencing the experiment, stock solution was freshly prepared and its concentration was analyzed using a HPLC. Algal inoculum was withdrawn from a chemostat operated under steady state and transferred into 300 ml, biochemical oxygen demand (BOD) test bottles together with dilution water (with growth medium) and test chemical. The BOD bottles were completely filled, with no headspace left. A water seal was provided to ensure a close-test environment. The BOD bottles were then placed on an orbital shaker operated at 100 rpm. Temperature and light intensity were kept at 24 ± 1°C and 65 µEm-2s-1 (±10%), respectively. After 48 hrs of test duration, the EC50 value was calculated. Probit analysis was used for determining the EC50 values. The population density of the algae was determined using an electronic particle counter. Based on the cell density of test organism Pseudokirchneriella subcapitata, the EC50 value was found to be 3.47 mg/l. Based on the EC50 value, chemical concluded as toxic and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

 

Above results supported by the studies from authoritative database 2018. Study was conducted to determine the effect of test chemical on the growth of algae. Test performed according to the OECD Guideline 201 (Alga, Growth Inhibition Test). Green algae exposed with the chemical for 72 hrs. After the 72 hrs of exposure with test chemical, effect were observed on the growth of algae. The EC50 (72 h) and NOEC value of the test substance 2,4,6-trichloroaniline to algae is observed to be 3.7 mg/L and 0.069 mg/L on the basis of growth rate parameter respectively. Thus considering the CLP Criteria for aquatic classification of the substance, it is concluded that 2,4,6-trichloroaniline exhibits short term toxicity to algae and is therefore classified as Aquatic Chronic category 2.

 

In the fifth study from secondary literature 2018 Evaluation of mode of action of test chemical 2,4,6-trichloroaniline on the growth of Haematococcus pluvialis. Based on the effect observed on the population rate, effect (EC10) were observed to be 12 mg/l.

 

Based on the overall studies from experimental report 2015, peer reviewed journal, authoritative databases and secondary sources 2018, it was concluded that the chemical 2,4,6-trichloroaniline concluded to be toxic to the aquatic algae and cyanobacteria and consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

 

 

Toxicity to microorganisms:

Based on the experimental data from various database for the target chemical study have been reviewed to determine the mode of action of2,4,6-trichloroaniline (CAS no. 634-93-5)on the growth and other activity of microorganisms. The studies are as mentioned below:

 

In the first key study from peer reviewed journal 2007, study was designed to access the effect of the substance 2,4,6-trichloroaniline on the luminescent bacteria Photobacterium phosphoreum carried out for 30 minutes. Before testing chemical was analyzed by HPLC. Based on the inhibition of luminescence of Photobacterium phosphoreum, the EC50 was observed at 4.61 mg/l. Based on the EC50, chemical can be consider as toxic.

 

First key study was supported by the second study from secondary sources 1987, 30 minutes study was conducted in which toxicity were measured on the basis of inhibition of luminescence of Pseudomonas putida. Based on the inhibition of cell multiplication after 30 minutes exposures, the EC10 of the test material 2, 4, 6-trichloroaniline is observed to be > 21 mg/L in Pseudomonas putida.

 

Similar study from same sources 30 minutes study was conducted in which toxicity were measured on the basis of inhibition of luminescence of Photobacterium phosphoreum. Toxicity effect of the test material 2, 4, 6-trichloroaniline was studied in Photobacterium phosphoreum. The toxicity effect were observed based on inhibition of luminescence during 30 minutes period. The EC50 is determine to be 4.60 mg/L.

 

Based on the above data, it was observed that the toxicity value ranges from 4.60 to > 21 mg/l.

 

Based on the overall studied of aquatic toxicity on the growth and morphological behavior of fishes, aquatic invertebrate, algae cyanobacteria and microorganisms, it was concluded that chemical 2,4,6-trichloroaniline (CAS no. 634-93-5) was toxic and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.