2-chlorotoluene

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-11-22 to 2001-03-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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 used to prepare the test solution was aerated before preparation and set to 20 ± 1 °C in a constant temperature bath. 46.2 μL (50 mg) of the test substance was collected, mixed with 50 mg of 2-methoxyethanol and 150 mg of HCO-40, diluted with purified water, and the volume was adjusted to 200 mL.

At the same time, a concentrated stock solution (HCO-40 750 mg/L, 2-methoxyethanol 250 mg/L) containing no test substance was prepared for the solvent stock solution.
Dilution water was filled in a 500 ml volumetric flask and the above concentrated test substance solution was added according to each concentration. The solvent stock solution was concentrated so that the solvent concentration stayed constant (8 mg/L) in all concentration groups. 100 mL each of final preparation was placed in 4 test vessels per concentration.

As solvent control diluted water containing only the test substance having the same concentration (8 mg/L) as the test concentration group, was used. Diluted water containing no test substance or solvent was used as the control group. The state (appearance) of the test solution at the time of preparation was colorless and transparent in all test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

1) Japanese name: Daphnia magna
2) Scientific name: Daphnia magna
3) Obtained from: National Environmental Research Institute, Environment Agency (currently National Environmental Research
Institute, Ministry of the Environment)
4) Obtained date: July 18, 1995
5) Sensitivity: Reference substance (Half-swimming inhibition concentration (EiC50) for 48 hours by potassium dichromate
(special grade reagent) = 0.68 mg/L (This value is the EiC50 value of 0.57 to approx. 1.02 mg/L since
June 1998 at our institute.)
6) Growth stage: Female neonate (within 24 hours)
7) Breeding conditions for parent Daphnia magna to obtain test organisms:
- Breeding water: Diluted water (see 3.2)
- Breeding density: 1 female/80 mL breeding water (25 females/2 L)
- Water temperature: 20 ± 1 °C
- Dissolved oxygen concentration: 60% or more of saturation concentration
- pH: 6.7 to approx. 8.5
- Lighting: Room light, 16 hours bright (800 lux or less)/8 hours dark
- Breeding period: January 17, 2001-January 31, 2001
- Parent mortality rate for 2 weeks
before start of exposure: <5%
- Occurrence of dormant
eggs and males: None
- Type of food: Chlorella vulgaris (unicellular green algae); Centrifuge the culture solution from living algae and replace it
with diluted water before use)
- Feed type: 0.2 mgC (organic carbon content)/daphnid/day
- Replacement of breeding water: Regularly exchanged 3 times/week

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

No data

Test temperature:

20.0 - 20.2 °C

pH:

7.9 - 8.1

Dissolved oxygen:

8.4 - 8.8 mg/L

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

Test concentration (n): 0.200, 0.360, 0.630, 1.10, 2.00 mg/L (common ratio: about 8)

Details on test conditions:

I. Test conditions
1) Exposure method: Semi-static (the entirely amount of test solution was replaced after 24 hours),
covered the water surface with Teflon sheet)
2) Exposure period: 48 hours
3) Test water volume: 100 mL
4) Number of test vessels: 4 test vessels per test concentration
5) Number of test organisms: 20 neonates/test concentration (5 neonates/per test concentration)
6) Test water temperature: 20+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 (800 lux or less)/8 hours dark
10) Feeding: No feeding

II. Dilution water
Use the M4 medium according Elendt (Appendix 1) of OECD Revised Guidelines No. 211.

III. Test concentration setting:
Based on the results of the preliminary test (2 test vessels each, 10 neonates/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: 0.200, 0.360, 0.630, 1.10, 2.00 mg/L (common ratio: about 8)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (special grade reagent)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

(95% confidence limit: 0.482 to 0.889 mg/L)

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.482 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

48 hours EiC50 value of 0.57 to approx. 0.68 mg/L

Table 2: Measured concentrations of the test substance in water (semi-static conditions)

Nominal concentration [mg/L]	Measured concentrations [mg/L]				Geometric mean during 24 hours [mg/L
	0 hour (new)	Percent of nominal	24 hours (old)	Percent of nominal
Control	< 0.0005	--	< 0.0005	---	---
Solvent Control	< 0.0005	--	< 0.0005	---	---
0.200	0.193	97	0.138	69	0.163
0.360	0.302	84	0.267	74	0.284
0.630	0.502	80	0.462	73	0.482
1.10	0.953	87	0.830	75	0.889
2.00	1.72	86	1.51	76	1.61

new: freshly prepared test solution

old: test solution after 24 hours of exposure

 

Table 3: The numbers if Immobile Daphnia (Percent Immobility)

 

Nominal concentration [mg/L]	Measured concentration [mg/L]	Cumulative numbers of immobilized Daphnia (Percent immobility)
		24 hours	48 hours
Control	---	0 (0)	0 (0)
Solvent Control	---	0 (0)	0 (0)
0.200	0.163	0 (0)	0 (0)
0.360	0.284	0 (0)	0 (0)
0.630	0.482	0 (0)	0 (0)
1.10	0.889	5 (25)	18 (90)
2.00	1.61	20 (100)	20 (100)

Validity criteria fulfilled:

yes

Conclusions:

The infuence on the immobility to daphnia was investigated in a semi-static GLP study following OECD 202. After 48 hours of exposure the EC50 value was 0.70 mg/L and the NOEC 0.482 mg/L by comparison of growth rate. The results relate to the geometric mean value of the measured value.

Executive summary:

This test is based on the OECD Guidelines for Testing of Chemicals No. It was conducted in accordance with 202 “ Daphnia, Swimming Inhibition Test and Reproductive Test” (1984)”. Acute immobilization test for Daphnia magna in o-chlorotoluene is performed. 50% immobilization inhibitory concentration (Eic50) and maximum no-effect concentration (NOECi) at 24 and 48 hours are determined under semi-static exposure conditions. After measuring the water temperature, dissolved oxygen concentration, and pH of the test solution, the neonates were added using a glass pipette, and that point was taken as the start of exposure. When the neonates were added, the total amount of breeding water in the pipette was kept within 1% of the test solution volume. Similarly, when the water was changed after 24 hours, the neonates were transferred to a new test solution using a glass pipette. The number of the immobilized neonates was observed 24 and 48 hours after the start of exposure. Water temperature, dissolved oxygen concentration and pH were measured at the start of exposure and before changing water for the test solution in one test vessel for each test group. Based on the results of the preliminary test the test concentrations of the main test were determined (0.200, 0.360, 0.630, 1.10, 2.00 mg/L (common ratio: about 8)). After 48 hours of exposure the EC50 value was 0.70 mg/L and the NOEC 0.482 mg/L by comparison of growth rate. The results relate to the geometric mean value of the measured value. In addition, as a result of an acute toxicity test using a reference substance (potassium dichromate, special grade reagent), the EiC50 for 48 hours was 0.68 mg/L. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute daphnia toxicity study.

Description of key information

EC50 (Daphnia magna; immob.; 48 h; semi-stat.; geom. mean meas.) = 0.70 mg/L (95% CI: 0.482 - 0.889)
NOEC (Daphnia magna; immob.; 48 h; semi-stat.; geom. mean meas.) = 0.482 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

0.7 mg/L

Additional information

The valid and reliable key study (MOE Japan, 2001) on acute toxicity of 2-chlorotoluol towards Daphnia magna was performed compliant with GLP according to OECD TG 202 (1984). Semi-static exposure conditions were applied, and test item concentrations analytically verified in fresh and aged (24 h) medium. The number of the immobilized neonates was observed 24 and 48 hours after the start of exposure. Water temperature, dissolved oxygen concentration and pH were measured at the start of exposure and before media exchange. Validity criteria of the guideline were met. Because measured concentration in aged media (after 24 h) were below 80% of nominal, effect concentrations were calculated based on geometric mean measured concentrations. The following results were obtained:
EC50 (Daphnia magna; immob.; 48 h; semi-stat.; geom. mean meas.) = 0.70 mg/L (95% CI: 0.482 - 0.889)
NOEC (Daphnia magna; immob.; 48 h; semi-stat.; geom. mean meas.) = 0.482 mg/L.

 

The reliable and relevant result of the key study is confirmed by not assignable (RL 4) data on acute aquatic toxicity towards Daphnia magna obviously determined in a flow-through system (no data on analytical confirmation).
The NOEC and EC50 for immobilization of Daphnia magna after 48 h (flow-through) are reported (secondary source) to be 0.45 mg/L (NOEC; 48h; immobilization; flow-through) and 1.1 mg/L (EC50; 48h; immobilization; flow-through) [Federal Register (USA) 1982, Vol. 47, No. 231].
These results are nearly equal to the results of the key study. While not assignable, these data corroborate the result of the key study.