Thymol

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 MAY 2005 - 14 OCT 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

1998

Deviations:

yes

Remarks:

Food was removed as a pre-treatment by centrifugation (3,000 rpm for 10 minutes) before measurement, but this operation was not described in the protocol. It is considered that there is no effect on the reliability of the test results.

Qualifier:

according to guideline

Guideline:

other: About the test method for investigating the toxicity of Class 3 monitoring chemical substances; Test on the effect on Daphnia breeding (Daphnia breeding test)

Version / remarks:

March 2004

Deviations:

yes

Remarks:

Food was removed as a pre-treatment by centrifugation (3,000 rpm for 10 minutes) before measurement, but this operation was not described in the protocol. It is considered that there is no effect on the reliability of the test results.

Principles of method if other than guideline:

About the test method for investigating the toxicity of Class 3 monitoring chemical substances
Test on the effect on Daphnia breeding (Daphnia breeding test)

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:

Sampling at the time of preparation and before water change.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Using the test stock solution in which the test sample was dissolved in test water by ultrasonic irradiation.
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 test stock solution is as follows:
from the start of exposure - after 6 days: 92.1 mg/L
from day 8 to day day 12: 96.3 mg/L
from day 14 to day 20: 90.0 mg/L

-Preparation of Standard solutions:
As the purity of the test substance is 100.0%, no purity correction was made in the preparation of the test stock solution. 50 mg of the test substance was weighed accurately on an electronic analytical balance and dissolved in acetonitrile to prepare a solution of 1000 mg/L of the test substance. This was diluted with acetonitrile to prepare a solution of 20.0 mg of the test substance that was further diluted with dechlorinated tap water to 1.0 mg/L. Further dilution to 0.1 mg/L with dechlorinated tap water resulted in the standard solution.

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
- Method of breeding: Breeding of larvae (14 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.
- Source: University of Sheffield, UK
- Feeding during test
- Food type: Chlorella vulgaris
- Amount: per Daphnia 0.1-0.2 mgC(organic carbon content)/day
- Frequency: once a day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

10 - 250 mg/L (CaCO3 equivalent)

Test temperature:

20 ± 1 °C
measured 19.9 - 20.0 °C

pH:

measured 7.6 - 8.1
variation was within 1.5

Dissolved oxygen:

during test minimum 60% or more of the saturation concentration (approximately 5 mg/L or more)
measured 8.9 - 9.0 mg/L

Salinity:

not applicable

Conductivity:

15.8 mS/m

Nominal and measured concentrations:

4.00, 2.00, 1.00, 0.25 mg/L (nominal) and control group

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): closed with lid
- Material, size, headspace, fill volume: 100 mL, filled with 80 mL test solution
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): once in 2 days
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water
- Total organic carbon: 1 mg/L
- Metals: not detected
- Pesticides:not detected
- Chlorine: < 0.02 mg/L
- Alkalinity: 20 mg/L
- Ca/mg ratio: 8.4/2
- Culture medium different from test medium: no
- Intervals of water quality measurement: The dissolved oxygen concentration, pH, water temperature and total hardness of the test solution were measured 6 times during the exposure period (at the time of preparation and before water change, repeated 3 times once a week). At the time of preparation, the test solution separated from the preparation container for water quality measurement was measured, and before the water was changed (one of the 10 test containers in each test group was measured). The light intensity was measured once during the exposure period.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light/8 hours dark
- Light intensity: ca 9.3 µE/m²s, light intensity below 15-20 μE/m²s by dark room light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Life and death of parent daphnia: Daphnia magna was considered dead when no movement of the limbs or abdomen was observed
- Number and condition of larvae produced: The number of larvae produced by the parent Daphnia magna was counted daily from the day of first birth to the end of the study. After counting and observation, the larvae were disposed of.
- Condition of the parent Daphnia magna: At the time of observation of the larvae, the size of the parent Daphnia magna, the presence or absence of abnormalities in swimming behaviour, the sex of the larvae, and the number of eggs in the nursery cell were examined. Also the presence or absence of dormant or aborted eggs were observed.

- Cumulative number of surviving offspring. The cumulative number of surviving litters (TF) and the mean cumulative number of surviving litters (TF) during the exposure period in each test vessel (per surviving parent) were calculated.

TEST CONCENTRATIONS
- Range finding study; Daphnia magna, 14d, semi-static
- Test concentrations: nominal 0.5, 1.58 and 5.0 mg/L and control (0 mg/L)
- Results used to determine the conditions for the definitive study: 40 % parental survival at 5.00 mg/L, 80 % parental survival at 1.58 mg/L
- 48-h acute inhibition test OECD 202; EC50 (48h): 4.46 mg/L, spacing factor 2, Concentrations: 4.00, 2.00, 1.00, 0.5 mg/L, 0.25 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (May, 2005)

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

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

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

3.49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

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

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

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

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: parent Daphnia magna

Remarks:

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

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Remarks on result:

other: no mortalities of parent daphnia occured, so it is the same for number of living offspring produced per surviving parental animal

Remarks:

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

Details on results:

HPLC analysis
The concentrations of the test substance in the test solutions measured during the exposure period were within ± 20% of the test concentrations, so the test concentrations were used to calculate the results.
- Concentration of test substance in test solution (paired value):
At the time of preparation 99.5 to 105%
Before water change 86.4-104%

- Number and rate of death of parent Daphnia magna
The cumulative mortality rate of the parent Daphnia magna in the control group and the concentration group was 0% at the end of the exposure, satisfying the criterion of not exceeding 20%.

- Date of first birth
The first calving date of the parent Daphnia magna in the control group was 9 days after the first calving date, which was considered to be in the appropriate range.　　　
In the 2.250-2.00 mg/L zone, the first calving day of the parent Daphnia magna was 9 days, the same as in the control group. In the 4.0 mg/L zone, the mean was 12.5 days, which was statistically significant compared with the control group.

- Average cumulative number of surviving litters
The average cumulative number of surviving litters per Daphnia magna in the control group over a 21-day period was 134, meeting the requirement of 60 or more litters. In the 0.250-2.00 mg/L zone, the mean cumulative number of surviving Daphnia magna per litter ranged from 126 to 136, which was not statistically significantly different from the control group. In the 4.0 mg/L zone, the mean number of surviving Daphnia magna was 49.1, which was statistically significantly different from the control group.

- Size and condition of the parent Daphnia magna
No symptoms were observed in the control group during the exposure period. In the 4.0 mg/L zone, growth inhibition was observed in all organisms from 3 days after exposure, but some organisms recovered to the same size as in the control zone after 19 days. In the 4.00 mg/L treatment, growth inhibition was observed in all organisms from 3 days after exposure.

- Occurrence of dormant eggs
No dormant eggs were found in any of the test plots during the exposure period. Mortality of larvae was observed in the control group and in the 2.0 and 4.0 mg/L treatments. Aborted eggs were observed in all test plots.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: When the test solution was prepared, it was clear and colorless in all test plots, and before the water change, it had a light green colour due to the feeding of Chlorella.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (48h) 0.283 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 0.231 ± 0.060 mg/L (n=49).

Reported statistics and error estimates:

EC50 survival
Using the calculated average cumulative number of surviving litters per surviving Daphnia magna in each test plot, the 50% reproductive inhibition concentrations (EC50) for 14 and 21 days were calculated by the Moving average method and their 95% confidence limits were also calculated.

Minimum effect concentration (LOEC) and maximum no effect concentration (NOEC)
The mean cumulative number of surviving litters per surviving parent Daphnia magna in each test site was tested for equality of variance using the Bartlett method, followed by a one-way analysis of variance. As significant differences were found in the analysis of variance, Dunnett's multiple comparison test was used.
The date of first birth in each test vessel was tested by Kruskal-Wallis rank sum test followed by Dunnett's multiple comparison method (non-parametric). The number of deaths of the parent Daphnia magna was not tested because no deaths were observed.

Validity criteria fulfilled:

yes

Conclusions:

The 21d-NOEC of thymol towards Daphnia magna was 2.0 mg/L. The EC50 (21d) was calculated to be 3.49 mg/L (95% confidence intervall: 3.27 - 3.78 mg/L)

Executive summary:

This study was carried out to determine the effect of the test substance thymol on reproduction in the test organism Daphnia magna according to OECD Guideline 211 (Daphnia magna Reproduction Test) and following GLP. The concentration of the test substance in the test solution was maintained within ±20% of the established concentration during the exposure period, and the environmental conditions were within the appropriate range, indicating that the test was appropriate in accordance with the test method.

Each daphnid was held in a separate vessel (10 vessels per concentration) and exposed under semi-static conditions for 21 days to concentrations of the test substance of nominal 4.00, 2.00, 1.00, 0.25 mg/L (nominal) and control group (0 mg/L). The 21d-NOEC of thymol towards Daphnia magna was 2.0 mg/L. The EC50 (21d) was calculated to be 3.49 mg/L (95% confidence intervall: 3.27 - 3.78 mg/L)

Description of key information

OECD Guideline 211 (Daphnia magna Reproduction Test); Daphnia magna, semi-static, 21 days, nominal concentrations: 4.00, 2.00, 1.00, 0.25 mg/L (nominal) and control group (0 mg/L); result: 21d-NOEC was 2.0 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 invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

2 mg/L