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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12/10/2015 - 24/10/2015
Reliability:
2 (reliable with restrictions)
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:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Principles of method if other than guideline:
This study was designed to assess the toxic effects of the test compound on the Zebra Fish. Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203.
GLP compliance:
not specified
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:
not specified
Vehicle:
yes
Details on test solutions:
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 48.89mg/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.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name:Zebra Fish
- Strain:Danio rerio
- Source: Asim Fancy Fish Aquarium, Dharampeth, Nagpur, MS (India)
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD):average 2.45 cm
- Weight at study initiation (mean and range, SD):0.149g (approx.)
- Method of breeding:
- Feeding during test
- Food type: Standard brand feed
- Amount: No data
- Frequency: once daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
75 mg of CaCO3
Test temperature:
21°C
pH:
7.8-8.2
Dissolved oxygen:
7.8-8.3 mg/l
Nominal and measured concentrations:
0.8 mg/L, 1.76 mg/L, 3.87mg/L, 8.5mg/L, 18.72mg/L, respectively
Details on test conditions:
- Test vessel: plastic aquaria
- Material, size, headspace, fill volume: water capacity of 5 liters
- Aeration: Aeration in test vessels was provided 1 day before the start of experiment
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 8 fishes
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:


OTHER TEST CONDITIONS
- Adjustment of pH: 7.4
- Photoperiod: 12 hours light and 12-hour darkness
- TDS (ppm): 382
- Conductivity (µScm-1): 1130
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 8.51 - < 18.72 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Experimental value
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
morphology
Remarks on result:
other: through probit analysis
Details on results:
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 mg/L (after rounding off)
Sublethal observations / clinical signs:

Mortality and symptoms

Nominal Concentration

(mg/l)

Number of fish

Mortality (cumulated) after

3 h

6 h

24h

48h

72h

96h

V1

V2

V1

V2

V1

V2

V1

V2

V1

V2

V1

V2

0 (control)

4

4

-

-

-

-

-

-

-

-

-

-

-

-

0.8 mg/L

4

4

-

-

-

-

-

-

-

-

-

-

-

-

1.76 mg/L

4

4

-

-

-

-

-

-

-

-

-

-

-

-

3.87 mg/L

4

4

-

-

-

-

-

-

-

-

-

-

-

-

8.51mg/L

4

4

-

-

  -

-

2

-

-

-

-

-

-

-

18.72 mg/L

4

4

2

3

1

-

1

1

-

-

-

-

-

-

Visible Symptoms

Nominal Concentration

(mg/l)

Number of fish

Mortality (cumulated) after

3 h

6 h

24h

48h

72h

96h

V1

V2

V1

V2

V1

V2

V1

V2

V1

V2

V1

V2

0 (control)

4

4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

0.8 mg/L

4

4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

1.76 mg/L

4

4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

3.87 mg/L

4

4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

NS4

8.51mg/L

4

4

IS4

IS4

IS4

IS4

IS2

IS4

NS2

NS4

NS2

NS4

NS2

NS4

18.72 mg/L

4

4

IS2

IS1

IS2

IS1

IS2

IS1

-

-

-

-

-

-

Explanation of symptoms:

A:apathy

N:Narcotic-like state

C:Swimming near water surface

M: Slow movements compared to control

NS:No abnormal symptoms 

S:Surfacing behaviour

L:Loss of equilibrium

SM:Shimming movement

H1:Hyperactivity

H2:Hypo activity

B:Bending behavior

F:Fin rot

IS:Invert Swimming

SB:Swimming behaviour

RF:Respiratory Function

P:Pigmentation

Validity criteria fulfilled:
yes
Conclusions:
Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for CAS No. 634-93-5 on Zebra fish (Denio rario) was determine to be >8.51mg/L & <18.72mg/L as experimental result and through probit analysis it was observed to be 10 mg/L.
Executive summary:

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.

Description of key information

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 FishDanio reriowere 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.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
10 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 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.