Thymol

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:

6 MAY 2005 - 20 SEPT 2005

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)

Version / remarks:

April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Test method for new chemical substances, etc.", Daphnia acute swimming inhibition test

Version / remarks:

2003

Deviations:

no

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

Analytical monitoring:

yes

Details on sampling:

The concentration of the test substance was measured at the beginning and at the end of exposure. 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: The test sample is dissolved in test water to prepare the stock solution
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 test sample was weighed and mixed with the test water and stirred for about 30 minutes with ultrasonic irradiation to prepare a stock solution of 100 mg/kg. The concentration of the test substance in the test stock solution was measured and the results were compared with the actual test solution. The measured concentration of the stock solution was 93.0 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: descendant of Daphnia magna (Clone A), subcultured in Kurume Plant
- Age at study initiation (mean and range, SD): juveniles within 24 hours of age were used
- Stage and instar at study initiation: Larvae
- Method of breeding: Breeding of larvae (27 days old) conditions same as test conditions: dechlorinated tap water, 20 +/- 1 °C, light-dark cycle (16 hours light/ 8 hours dark). A group with 100% survival was used for the test.
- Feeding during breeding: Chlorella vulgaris, 0.1 -0.2 mgC
- Source: Sheffiled University in the United Kingdom
- Age of parental stock (mean and range, SD): 27 days
- Feeding during test : no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

38.8 mg/L

Test temperature:

20 +/- 1°C
measured: 19-21°C

pH:

7.3
measured: 7.5 - 7.7

Dissolved oxygen:

during test: minimum 60 %
measured: 8.9 - 9.0 mg/L

Salinity:

not applicable

Conductivity:

15.8 mS/m

Nominal and measured concentrations:

8.00, 5.71, 4.08, 2.92 and 2.08 mg/L and control (0 mg/L) nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): closed with lid
- Volume of solution: 100 mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): no change of test solution
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

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 mg/L
- 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 start and end of exposure. At the start of exposure, the test solution separately separated is measured, and at the end of exposure, each test group. The measurement was performed on one of the four test containers.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light/ 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
swimming inhibition

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4

RANGE-FINDING STUDY
48h, 20 animals per concentration first test
48h, 5 animals per concentration second test
- Test concentrations:
1.2, 2.04, 3.46, 5.88, 10 mg/L first test
1.98, 2.96, 4.44, 6.67, 10 mg/L second test
- Results used to determine the conditions for the definitive study: The test concentrations and spacing factor were determined from the preliminary test results.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (May, 2005)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% confidence limit: 4.07 - 4.88 mg/L; the test substance concentrations were confirmed to be stable within +/- 20% of initial nominal concentration via analytical measurement.

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

2.92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

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

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

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:
Test substance concentration in test solution (paired value):
111 and 101% at the start of exposure
106 and 96.1% at the end of exposure
The concentrations were maintained within ± 20% of the set values (nominal values)

- Behavioural abnormalities: During the exposure period smptoms observed were lethargy, swimming inhibition, and decreased activity
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, the test solution was clear and clolourless throughout the whole exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No, the test solution was clear and clolourless throughout the whole exposure period.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (48h) 0.2 mg/L
This value is within the specified range of the background data at the Kurume site (mean ± 2 x standard deviation).
The mean ± standard deviation was 0.232 ± 0.060 mg/L (n=48).

Reported statistics and error estimates:

EC50 was calculated by Probit method. Since the measured test substance concentration was within ± 20% of the set test concentration (nominal), it was set to calculate the result.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (48h) of thymol towards Daphnia magna was 4.46 mg/L with a 95% confidence limit of 4.07 to 4.88 mg/L.

Executive summary:

This acute immobilisation test was conducted to determine the acute toxicity of thymol dissolved in water towards aquatic invertebrates (Daphnia magna) according to OECD Guideline 202 and according to the Daphnia acute swimming inhibition test as prescribed in "Methods for testing new chemical substances, etc." following 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.

Daphnia magna were exposed for 48 hours at static conditions to nominal concentrations of the test substance thymol of 8.00, 5.71, 4.08, 2.92 and 2.08 mg/L and control (0 mg/L) in quadruplicate (20 daphnids per concentration). Symptoms observed during the exposure period were lethargy, swimming inhibition, and decreased activity. The EC50 (48h) was determined to be 4.46 mg/L with a 95% confidence limit of 4.07 to 4.88 mg/L.

Description of key information

OECD 202, GLP, Daphnia magna, 48 hours, static, nominal concentrations : 8.00, 5.71, 4.08, 2.92 and 2.08 mg/L and control (0 mg/L) in quadruplicate (20 daphnids per concentration). Symptoms observed during the exposure period were lethargy, swimming inhibition, and decreased activity. The EC50 (48h) was determined to be 4.46 mg/L with a 95% confidence limit of 4.07 to 4.88 mg/L. The test substance concentrations were confirmed to be stable within +/- 20% of initial nominal concentration via analytical measurement.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

4.46 mg/L

Additional information

The most sensitive acute toxicity of thymol to invertebrates was obtained in a multispecies test according to Methods of US-EPA, 1975 (Ewell, 1986). Simultaneously seven species from five phyla were exposed to the test item concentrations of nominal 0.1, 1.0, 10 and 100 mg/L in the multi-species test. A duplicate single species test was also performed with thymol.

In this multispecies test, a 96 h LC50 of 3.2 mg/L of thymol to Daphnia magna was obtained.

Due to deviations from standard guidelines for acute toxicity to daphnia 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.