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Ecotoxicological information

Short-term toxicity to fish

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Link to relevant study record(s)

Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-11-22 to 2001-03-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
High Performance Liquid Chromatography (HPLC)
Details on sampling:
For each test vessel in all test groups, 0.75 mL of each test solution before and after water change was taken three times during the exposure period, equal amounts of acetonitrile were added, mixed, and then analyzed by HPLC. The test substance of each test solution was quantified from the ratio with the peak area of the standard solution.
Vehicle:
yes
Remarks:
2-methoxyethanol and HCO-60
Details on test solutions:
The diluted water at the time of preparing the test solution was set to 24 ± 1 °C in a constant temperature bath before preparation. To 462 μL of the test substance (500 mg in terms of specific gravity), 500 mg of 2-methoxyethanol and 1500 mg of the dispersion aid (HCO-40) were added, mixed and dissolved, and then the volume was adjusted to 500 mL with purified water to prepare the test substance stock solution. The test substance stock solution has a concentration of 1000 mg/L.
As for the control solution of the solvent agent, 500 mg of 2-methoxyethanol and 1500 mg of the dispersion solvent agent (HCO-40) were added to 500 mL of purified water.
The test substance stock solution and the solvent agent stock solution were collected, and the volume was adjusted to 5.0 L with diluted water. amounts are listed in Table 1 (see. additional information) .
The test solution at the time of preparation was colorless and transparent in all test groups.
Test organisms (species):
Oryzias latipes
Details on test organisms:
1) Japanese name: Himedaka
2) Scientific name: Oryzias latipes
3) Obtained from: Sankyo Fisheries Co., Ltd. (1-1, Ichigayatamachi, Shinjuku-ku, Tokyo)
4) Date of aquisition: February 5, 2001
5) Lot number: 01-H-0205
6) Body length: 2.21 cm (1.97 to 2.45 cm), n = 10
7) Weight: 0.148 (0.103 to 0.244 g), n = 10
8) Sensitivity: 96 hours median lethal concentration (LC50) = 0.86 mg/L
(this value is the LC50 value 0.44 - 1.5 mg/L since 1995) with the reference substance (copper sulfate (H)
pentahydrate, reagent technical grade), n = 14)
9) Acclimatisation period: From February 9, 2001 to February 26, 2001, the mortality rate was less than 5% for 7 days before the start
of exposure, and macroscopically healthy and normal individuals were used in the study.
10) Holding conditions:
- Holding water: diluted water (see 3.2)
- Holding condition: Water type (breeding density: 1.0 g/L/day or less)
- Water temperature: 24 ± 1 ° C
- Dissolved oxygen concentration: 80% or more of saturation concentration
- pH: 6.5 to 8. 5
- Lighting: Room light, 16 hours light (1000 lux or less)/8 hours dark
- Food type: Tetrabike Tetramine
- Feeding amount: Approximately 2% of fish body weight/day (no feeding from 24 hour before the start of exposure)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
No data
Test temperature:
23.3 - 23.4 °C
pH:
6.9 - 7.5
Dissolved oxygen:
5.5 - 8.4 mg/L
Salinity:
n.a.
Conductivity:
No data
Nominal and measured concentrations:
Test concentration (n): 0.25, 4.40, 7.90, 14.0, 25.0 mg/L (common ratio: about 1.8)
Details on test conditions:
I. Test conditions
1) Exposure method : Semi-static (the entire amount of test solution was replaced after 24 hours),
cover the water surface with Teflon sheet
2) Exposure period : 96 hours
3) Test water volume : 5.0 L/test aquarium
4) Number of test vessels : 1 aquarium/test design
5) Number of test organisms : 10 fish/test design
6) Test water temperature : 24+1 °C
7) Dissolved oxygen concentration : 60% or more of saturation concentration
8) pH : No adjustment of pH of test solution
9) Illumination : 16 hours bright (1000 lux or less)/8 hours dark
10) Feeding : No feeding

II. Diluted water
Dechlorinated water (Yokohama city tap water treated with activated carbon to remove residual chlorine, etc., and well ventilated) was used.

III. Test concentration design
Based on the results of the preliminary test (1 test vessel each, 5 fish/test concentration) shown in the table below, the test concentration of the main test was determined as follows.
Test concentration: control group, solvent control group: 0.25, 4.40, 7.90, 14.0, 25.0 mg/L (common ratio: about 1.8)

IV Observation (Toxicological symptoms):
- Death : A case in which no reaction is observed when a stimulus is given.
- Abnormal respiration : The movement of the gill lid is different from that of the fish in the control group.
- Abnormal swimming : Swimming that is clearly different from the fish in the control area. Slow movement,
irritability, convulsions, inversion, etc.
- Inability to swim : Those who are moving at the bottom or on the surface of the water but are unable to swim in the water.
Includes rollover and asphyxia.

V. Water temperature, dissolved oxygen concentration and pH of the test solution:
The pH, dissolved oxygen concentration and water temperature were measured once daily during the exposure period.
Reference substance (positive control):
yes
Remarks:
copper sulfate (H) pentahydrate, reagent technical grade
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
7.67 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other:
Remarks:
(95%-confidence limit: 5.66 mg/L to 10.4 mg/L)
Results with reference substance (positive control):
96 hours median lethal concentration (LC50) = 0.86 mg/L
Sublethal observations / clinical signs:

Table 2: Measured concentrations of substance in test water

 Nominal concentration 

Measured concentration [mg/L]
(Percent of nominal)    
 Mean* measured concentration
[mg/L]   
 0 hour (new)  24 hours (old)
 Control (Blank)  < 0.003  < 0.003  ---
Solvent Control  < 0.003  < 0.003  ---
 2.50  2.40
(96)
 2.14
(86)
 2.27
(91)
 4.40  4.11
(93)
 3.76
(85)
 3.93
(89)
 7.90  7.13
(90)
 6.33
(80)
 6.72
(85)
 14.0  13.0
(93)
 12.0
(86)
 12.5
(89)
 25.0  23.2
(93)
 --
(--)
 23.2
(93)

* geometric mean
new: freshly prepared test solutions
old: test solutions after 24hours exposure
--: No measurement was made because all Medaka were dead at this observation time.

Table 3: Mortality of the Medaka (Oryzas latipes) exposed to the test substance

 Nominal concentration [mg/L]     Mean* measured concentration [mg/L]     cumulative mortality (Percent mortality)         
 24 hours  48 hours  72 hours   96 hours 
 Control  ----  1 (10)  1 (10)  1 (10)  1 (10)
Solvent Control  ----  0 (0)  0 (0)  0 (0)   0 (0)
 2.50  2.27  0 (0)  1 (10)  1 (10) 1 (10) 
 4.40  3.93  0 (0)  0 (0)  1 (10)  1 (10)
 7.90  6.72  0 (0)  0 (0)  2 (20)  4 (40)
 14.0  12.5  8 (80)  8 (80)  8 (80)  9 (90)
 25.00  23.2  10 (100)  10 (100)  10 (100)  10 (100)

* geometric mean

Validity criteria fulfilled:
yes
Conclusions:
The mortality to fish was investigated under semi-static static conditions in a GLP study following OECD 203. After 96 hours exposure, the median lethal concentration (LC50) based on nominal concentration was 7.7 mg/L.
Executive summary:

This test is based on the OECD Guidelines for Testing of Chemicals No. It was conducted in accordance with 203 “Fish Toxicity Test" (1992). For o-chlorotoluene, an acute toxicity test against medaka (Oryzias latipes) was performed to determine the median lethal concentration (LC50) for 96 hours under semi-static exposure conditions. At the start of exposure, the weight and scale length of 10 fish acclimatized in the same tank as the test fish were measured. No feeding was done during the dew. After measuring the pH, dissolved oxygen concentration, and water temperature of the test solution in each test container, 10 fish under test were placed in the test container per concentration group. The pH, dissolved oxygen concentration and water temperature were measured once daily during the exposure period. Deaths were recorded 24, 48, 72 and 96 hours after the start of exposure and any signs or abnormalities of toxicity observed were recorded. Other specific cases (bending of the back, bleeding, change in body color, secretion of mucus, etc.) are described separately when observed. When dead individuals were found, they were promptly removed to prevent deterioration of water quality. Based on the results of the preliminary test the test concentration of the main test were determined (0.25, 4.40, 7.90, 14.0, 25.0 mg/L (common ratio: about 1.8)) Recovery rates for the substance were > 80 % of the nominal concentration in all sampling points.. After 96 hours exposure, the median lethal concentration (LC50) based on nominal concentration was 7.7 mg/L. In addition, as a result of an acute toxicity test using a reference substance (copper sulfate (H) pentahydrate, reagent special grade) of the test fish, the LC50 for 96 hours was 0.86 mg/L. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute fish toxicity study.

Description of key information

The acute toxicity of 2-chlorotoluene towards fish was tested following the OECD Guideline 203, yielding a LC50 (96h; Oryzias latipes; analytically confirmed) of 7.7 mg/L (MOE Japan, 2001).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC50
Effect concentration:
7.67 mg/L

Additional information

The valid and reliable key study (MOE Japan, 2001) was performed compliant with GLP according to OECD TG 203 (1992). 2-chlorotoluene toxicity towards Japanese Medaka (Oryzias latipes) was assessed for 96 hours under semi-static exposure conditions. 10 fish were placed in the test container per concentration group. The pH, dissolved oxygen concentration and water temperature were measured once daily during the exposure period and validity criteria of the testing guideline were met. Deaths were recorded 24, 48, 72 and 96 hours after the start of exposure and any signs or abnormalities of toxicity observed were recorded. Analytical monitoring of test item concentrations in fresh and aged (24 h) medium confirmed the nominal concentration (within +/- 20%). Accordingly, evaluation of results was performed based on nominal concentrations.
Results of the study:
LC50 (Oryzias latipes; 96 h; analytically confirmed) = 7.67 mg/L (95% CI: 5.66 - 10.4).


The reliable and relevant result of the key study is confirmed by not assignable (RL 4) data on acute aquatic toxicity towards Pimephales promelas and Oncorhynchus mykiss, which obviously were determined in a flow-through system with analytically confirmed exposure concentrations (Federal Register USA, 1982) with the following results:
LC50 (Pimephales promelas; 96 h) = 7.5 (95% CI: 6.1-9.8);
LC50 (Oncorhynchus mykiss; 96 h) = 2.3 mg/L (95% CI: 1.8-3.0).
These results are very close to and of the same order of magnitude as the result of the key study. While not assignable, these data corroborate the result of the key study.