Thymol

Short-term toxicity to fish

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:

16 MAY 2005 - 21 SEPT 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Fish acute toxicity test as prescribed in "Methods for testing new chemical substances, etc." Acute toxicity test to fish

Deviations:

no

Principles of method if other than guideline:

Fish acute toxicity test as prescribed in "Methods for testing new chemical substances, etc." Acute toxicity test to fish

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 100 %
Water solubility: 1g/L
Dissolved in ethanol, chloroform, diethyl ether, olive oil, glacial acetic acid
Stability: varies with light

Analytical monitoring:

yes

Details on sampling:

The concentration of the test substance was measured at the beginning of exposure, before and after water change and at the end of exposure. The test solutions were mixed from equal volumes of the middle layer of each container. The concentration of the test substance was analysed by high speed liquid chromatography (HPLC).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solution made by dissolving test sample in test water by ultrasonic irradiation
As the test substance has a purity of approximately 100.0%, no purity correction was made in the preparation of the test stock solution. The required amount of the test substance was weighed, mixed with test water, sonicated for about 10 minutes and stirred.
After sonication the mixture was stirred to prepare a 100 mg test stock solution. The concentration of the test substance in the stock solution was measured and the result was compared with the actual concentration of the test substance. The measured concentration of the stock solution was 91.6 mg/L.

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: Rice fish, Japanese medaka
- Source: Nakajima fish farm
- Age at study initiation (mean and range, SD): 10 months
- Length at study initiation (length definition, mean, range and SD): 2.3 +/- 1.2 cm

ACCLIMATION
- Acclimation period: 30 days
- Acclimation conditions (same as test or not): running tap water
- The organisms were bathed with oxytetracycline and NaCl at the time of acquisition and acclimatisation
- Type and amount of food during acclimation: carp formula, 2-4%
- Feeding frequency during acclimation: day 2 to 5
- Health during acclimation (any mortality observed): mortality rate 7 days before the test was 0.38 %

FEEDING DURING TEST (as applicable)
- none, stopped 24 hours before the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

38.8 mg/L

Test temperature:

24 ± 1 °C
measured 23.5 - 24.8 °C

pH:

7.3
measured 7.1 - 7.7

Dissolved oxygen:

during acclimatisation: 80 %
during test: minimum 60 %
measured: 6.3 - 8.7 mg/L

Salinity:

not applicable

Conductivity:

15.8 mS/m

Nominal and measured concentrations:

10, 7.14, 5.10, 3.64 and 0.911 mg/L and control (0 mg/L) nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: glass container
- Type (delete if not applicable): closed with lid
- Material, size, headspace, fill volume: 3L, filled with 2.5 L test solution
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): once in two days (entire amount of test solution was replaces 48 hours after the start of exposure)
- No. of organisms per vessel: 4
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water
- Total organic carbon: 0.3 mg/L
- Metals: not detected
- Pesticides: not detected
- Chlorine: < 0.02 mgL
- Alkalinity: 36 mg/L
- Ca/mg ratio: 11/2.8
- Culture medium different from test medium: no
- Intervals of water quality measurement: The dissolved oxygen concentration, pH and water temperature of the test solution were measured at the beginning of exposure, before and after water change, and at the end of exposure. Water quality was also measured at 24 and 72 h. For the concentrations at which all individuals died (7, 14 and 10.0 mg/L), water quality and dissolved oxygen was measured at 96 and 24 h after all individuals were confirmed dead.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light/ 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Analysis of test substance concentration (HPLC): at the beginning of exposure, before and after water change, and at the end of exposure
- Death and symptoms were observed at 3, 24, 48, 72 and 96 hours after the start of exposure. Fish were considered dead if there is no movement (proboscis, gill lid movement etc.) and there is no reaction by lightly touching the caudal peduncle with a glass rod. Dead individuals should be removed promptly.
- After the end of exposure, the total length and body weight of the test organisms were measured

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4 and 4 (0.911 mg/L)
- Range finding study
semi static, no aeration, 2 fish/1 L at first test and 4 fish/2.5L at second test
- Test concentrations: first test: 1, 2.6, 5.1, 10 mg/L; second test: 1 and 2 mg/L
- Results used to determine the conditions for the definitive study: In the preliminary study, it was confirmed that the concentration of the test substance in the test solution was stable during the exposure period. Therefore, the concentration of the test substance in the test solution was measured in the highest and lowest concentration zones.

Reference substance (positive control):

yes

Remarks:

copper(n) sulphate pentahydrate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

95% confidence limit: 3.89 to 5.53 mg/L; the test substance concentrations were confirmed to be stable within +/- 20% of initial nominal concentration via analytical measurement.

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

7.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

The test substance concentrations were confirmed to be stable within +/- 20% of initial nominal concentration via analytical measurement.

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.911 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

The test substance concentrations were confirmed to be stable within +/- 20% of initial nominal concentration via analytical measurement.

Details on results:

HPLC analysis:
Concentration of the test substance in the test solution (paired values):
The concentration of the test substance in the test solution was 83.8-102.8 mg/L at the beginning of the exposure and after the water change.
The measured concentrations of the test substance in the test solution were 83.8% to 102% of the set value at the beginning of exposure and after water change, and 92.0% to 99.3% at the end of exposure and before water change. The concentrations were maintained within ± 20% of the set values (nominal values).

- Observations on body length and weight: Size [mean ± standard deviation (n = 8)1
　　　Total length 2.7 ± 0.089 cm
　　　Weight 0.18 ± 0.012g
- Other biological observations: Symptoms observed during the exposure period were superficial concentration, loss of equilibrium, lethargy and decreased activity.
- Mortality of control: none
- Other adverse effects control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, the solution was always colourless and clear
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: yes
- LC50: LC50 (96h) = 0.369 mg/L
This value is within the specified range of the background data at the Kurume site (mean ± 2 x standard deviation: 0.111 - 1.02 mg/L).
The mean ± standard deviation was 0.568 ± 0.228 mg/L (n=31).

Reported statistics and error estimates:

For LC50 calculation Binomial method for 24 hours, Probit method for 48, 72 and 96 hours was used.

Validity criteria fulfilled:

yes

Conclusions:

The LC50 (96h) of thymol towards the Japanese medaka was 4.67 mg/L with a 95% confidence limit of 3.89 to 5.53 mg/L.

Executive summary:

This test was conducted to determine the acute toxicity of thymol dissolved in water towards the Japanese medaka (Oryzias latipes) according to OECD Guideline 203 and the Fish acute toxicity test as prescribed in "Methods for testing new chemical substances, etc." and in compliance with GLP criteria. The nominal concentration of the test substance in the test solution was kept within ± 20% of the set concentration and the environmental conditions were within the appropriate range. 4 fish per vessel were exposed under semi-static conditions for 96 hours to concentrations of the test substance of 0, 10, 7.14, 5.10, 3.64 and 0.911 mg/L in duplicate (8 fish per concentration). Symptoms observed during the exposure period were superficial concentration, loss of equilibrium, lethargy and decreased activity. The LC 50 (96h) was determined to be 4.67 mg/L with a 95% confidence limit of 3.89 to 5.53 mg/L.

Description of key information

OECD 203, GLP, Oryzias latipes, 96 h, nominal concentration: 0, 10, 7.14, 5.10, 3.64 and 0.911 mg/L in duplicate (8 fish per concentration). Symptoms observed during the exposure period were superficial concentration, loss of equilibrium, lethargy and decreased activity. The LC50 (96h) was determined to be 4.67 mg/L with a 95% confidence limit of 3.89 to 5.53 mg/L; the test substance concentrations were confirmed to be stable within +/- 20% of initial nominal concentration via analytical measurement.

(MITI, 2005).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

4.67 mg/L

Additional information

Another acute toxicity value of thymol to fish was obtained in a multispecies test according to Methods of US-EPA, 1975 (Ewell, 1986).

Pimephales promelas was exposed to concentrations of nominal 0.1, 1.0, 10 and 100 mg/L. A duplicate single species test was also performed with thymol. The 96 h LC50 of thymol was 3.2 mg/L. Due to deviations from standard guidelines for acute toxicity to fish such as less than 5 test concentrations, missing analytics and protocol, the LC50(96h) value has not been chosen as key value for risk assessment but it does strongly support the key value.