2,4,6-trichloroaniline

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/10/2015 to 19/10/2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Experimental test result performed using standard OECD test guidelines

Justification for type of information:

Experimental test result performed using standard OECD test guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

This study was designed to access the effect of the substance 2,4,6-trichloroaniline on the growth of green alga Chlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201 – Alga growth inhibition test”.

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,4,6-trichloroaniline
- Molecular formula (if other than submission substance): C6-H4-Cl3-N
- Molecular weight (if other than submission substance): 196.464
- Smiles notation (if other than submission substance): c1(c(cc(Cl)cc1Cl)Cl)N
- InChl (if other than submission substance): 1S/C6H4Cl3N/c7-3-1-4(8)6(10)5(9)2-3/h1-2H,10H2
- Substance type: organic

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

The test solution was prepared in aseptic condition. 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.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): National Environmental Engineering Research Institute (NEERI), Nagpur (Laboratory)
- Method of cultivation: Bold’s Basal Medium(BBM)

ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

24 °C ±2°C.

pH:

6.3-6.8

Nominal and measured concentrations:

0.5 mg/l, 1.25 mg/l, 3.125mg/l, 7.813 mg/l, 19.531 mg/l and 48.828mg/l. All the six concentration were in geometric series spaced by a factor of 2.5

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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.
- Chlorophyll measurement: No data
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Test concentrations: Six test concentration were: 0.5 mg/l, 1.25 mg/l, 3.125mg/l, 7.813 mg/l, 19.531 mg/l and 48.828 mg/l
- Results used to determine the conditions for the definitive study: Mortality of test organisms

Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 24°C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.445 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated from equation through probit analysis

Details on results:

The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flasks cells appeared shrinked and bubble like structures were observed in the cells.

Reported statistics and error estimates:

To obtain a quantitative concentration-response relationship by regression analysis,alinearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Table 1: Showing the average cell count using Haemocytometer of the experimental flasks at an equal interval of 24hrs, 48hrs and 72hrs

Experimental Flasks and test concentration	24 Hours	48 Hours	72 Hours
Control
Replicate 1	51X 10^4	81.5X 10^4	100X 10^4
Replicate 2	49.5X 10^4	78.5X 10^4	102.5X 10^4
Replicate 3	57X 10^4	72X 10^4	107.5X 10^4
CAS No. 634-93-5
0.5 mg/l
Replicate 1	37X 10^4	32X 10^4	27X 10^4
Replicate 2	36.5X 10^4	33.5X 10^4	20X 10^4
1.25 mg/l
Replicate 1	31X 10^4	31X 10^4	19X 10^4
Replicate 2	30X 10^4	35.5X 10^4	19.5X 10^4
3.125 mg/l
Replicate 1	24X 10^4	20.5X 10^4	21.5X 10^4
Replicate 2	24.5X 10^4	20.5X 10^4	16X 10^4
7.813 mg/l
Replicate 1	21X 10^4	16.5X 10^4	9.5X 10^4
Replicate 2	23.5X 10^4	13.5X 10^4	9X 10^4
19.531 mg/l
Replicate 1	16X 10^4	9.5X 10^4	6.5X 10^4
Replicate 2	18.5X 10^4	10.5X 10^4	7X 10^4
48.828 mg/l
Replicate 1	13.5X 10^4	6.5X 10^4	3.5X 10^4
Replicate 2	13X 10^4	5.5X 10^4	1X 10^4

Table 2 : Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

	CONTROL		0.5mg/l		1.25mg/l		3.125mg/l		7.813mg/l		19.531mg/l		48.828mg/l
Average Specific Growth rate (µ )	R1	1.535	R1	1.099	R1	0.981	R1	1.023	R1	0.750	R1	0.624	R1	0.418
	R2	1.543	R2	0.999	R2	0.990	R2	0.924	R2	0.732	R2	0.649	R2	0
	R3	1.559
Mean of Avg. Specific growth rate	1.546		1.049		0.986		0.973		0.741		0.636		0.209
Percentage Inhibition (%I)	_		32.166		36.228		37.028		52.038		58.839		86.493

Table 3: Depicting pH values at test initiation (0 Hours) and test termination ( 72 Hours)

Experimental Flasks and test concentration	0 Hours	72 Hours
CONTROL
Replicate 1	6.3	6.8
Replicate 2	6.3	6.7
Replicate 3	6.3	6.8
CAS No. 634-93-5
0.5mg/l
Replicate 1	6.4	6.7
Replicate 2	6.4	6.7
1.25mg/l
Replicate 1	6.4	6.7
Replicate 2	6.4	6.7
3.125mg/l
Replicate 1	6.3	6.8
Replicate 2	6.3	6.8
7.813 mg/l
Replicate 1	6.4	6.7
Replicate 2	6.4	6.7
19.531mg/l
Replicate 1	6.4	6.5
Replicate 2	6.4	6.5
48.828 mg/l
Replicate 1	6.4	6.3
Replicate 2	6.4	6.3

Validity criteria fulfilled:

yes

Conclusions:

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.

Executive summary:

The 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.

Description of key information

The 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.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.445 mg/L

Additional information

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 67.5 mg/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 10000cells/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.