Thymol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 JUN 2005 - 12 OCT 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan; Test Methods for New Chemical Substances: Algae growth inhibition test

Version / remarks:

2003

Deviations:

no

Principles of method if other than guideline:

Also taken into account:
OECD Guidance Document 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures" (September 2000)
OECD Guidelines for Testing of Chemicals - Draft Guideline 201 (April 2004)

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 in the test solution was determined at the beginning and end of the exposure. The test solution for the measurement at the start of exposure was taken from the preparation container. The test solution at the end of the exposure period was mixed with an equal volume of the test solution from each test container and centrifuged (3000 rpm for 10 min) to remove the algae. 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: Preparation of the test solution: The test solution was prepared by dissolving the test sample in the medium and filtering it by suction.
As the purity of the test substance is 100.0 %, no purity correction was made in the preparation of the test stock solution.
The required amount of the reagent was weighed, added to the medium and dissolved by stirring for about 1 hour to prepare a solution of 100 mg/L. The solution was filtered by suction through a membrane filter 0.22 μm (Nihon Millipore Co., Ltd.) and the filtrate was used as the test solution. The required amount of test solution and medium was mixed in a preparation vessel, stirred and divided into test vessels.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection (12301 Parklawn Drive Rockville, Maryland)
- Age of inoculum (at test initiation): passive culture

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 to 24°C (± 2°C)
measured: 23.2 - 23.6 °C

pH:

beginning of exposure: 7.5 - 7.8
end of exposure: 7.5 - 7.7

Salinity:

not applicable

Nominal and measured concentrations:

nominal 20.0, 9.09, 4.13, 1.88, 0.854 mg/L and control

Details on test conditions:

TEST SYSTEM
- Test vessel: Flask
- Type (delete if not applicable): closed , ventilated SILICOSEN lid
- Material, size, headspace, fill volume: Sterile glass triangular flask of 500 mL capacity, filled with 100 mL
- Aeration: continous shaking incubation (100 times/minute)
- Initial cells density: 10E4 cells/mL (The pre-culture medium containing cells in the logarithmic growth phase was inoculated into the test medium at a concentration of 10E4 cells/mL.)
- Control end cells density: 121 x 10E4 cells/ mL
- No. of vessels per concentration (replicates): 3 x 100 mL
- No. of vessels per control (replicates): 6 x 100 mL

GROWTH MEDIUM
- Standard medium used: yes, according to Draft Guideline 201 (April 2004) of the OECD Guidelines for the Testing of Chemicals

TEST MEDIUM / WATER PARAMETERS
OECD medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the test solution was measured at the beginning and at the end of the exposure. The pH of the test solutions was measured at the beginning and at the end of the exposure period, for the test solutions taken separately at the beginning of the exposure period and for one test vessel per test plot at the end of the exposure period. Temperature and light intensity in the incubator were measured once a day during the exposure period using a glass-electrode hydrogen ion meter (HM-14P, Toa-DK) for pH, a calibrated glass rod thermometer for temperature, and a portable light intensity meter (QSL-100, Biospherical Instruments) for light intensity.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination with fluorescent lighting (400 - 700 nm)
- Light intensity and quality: 60-120 μE/m²/s (variation range ± 20%), measured: 104 - 116 μE/m²/s.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber (cell concentration), every 24 hours up to 72 hours after the start of exposure. In order to measure the background of the test solution, the culture medium was measured at the same time and corrected for blanks. At the end of the exposure, one test vessel per test section was examined under a biological microscope (BX41, Olympus).
- Chlorophyll measurement: no
- Other: The condition of the test solution was observed at the beginning and end of the exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study ;
First test: The test solution is prepared by mixing the medium and the test substance, stirring for about 1 hour and then dissolving.
Second test: The test solution was prepared by mixing the medium with the test substance, stirring for about 1 hour, dissolving, and then filtering through a 0.22 µm membrane filter.
- Test concentrations: first test: 0.3, 1.0, 3.0, 10. , 30.0 mg/L; second test: 0.854, 1.88, 4.13, 9.09, 20.0 mg/L

CULTURING APPARATUS
-Details on culturing apparatus used:
A device capable of maintaining temperature, continuous illumination and continuous shaking, as well as maintaining a constant light intensity (rotary shaking incubator with a low-temperature thermostatic bath, TB-C-50RL, manufactured by Takasaki Scientific Instruments) was used.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit; not calculated

Remarks:

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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limit: 4.84 to 12.4 mg/L

Remarks:

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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other:

Remarks:

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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

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: Concentration of test substance (vs. set value):
93.6-101% at the beginning of exposure
86.7-92.3% at the end of exposure
As the concentrations of the test substance in the test solutions measured during the exposure period were within ±20 % of the test concentrations, the nominal test concentrations were used to calculate the results.

- Exponential growth in the control (for algal test): yes; The growth of the test algae in the control group showed a logarithmic growth until the end of the exposure. At the end of the exposure, the growth of the test algae was more than 114 times higher than the initial cell number, meeting the efficacy criterion (more than 16 times higher growth). The average daily growth rates in the control group were 1.61, 1.78 and 1.40 for the 0-1, 1-2 and 2-3 days, respectively, with a mean and standard deviation of 1.60 ± 0.1 g per day. The mean and standard deviation of the daily growth rates were 1.60 ± 0.1 g. The calculated coefficient of variation was 11.5%, which met the efficacy criterion (not to exceed 35%). The mean and standard deviation of the growth rate between repetitions in the control group was 1.61 ± 0.02 for 0-1 days, 1.78 ± 0.10 for 1-2 days and 1.40 ± 0.08 for 2-3 days. The calculated coefficients of variation were 1.19, 5.39 and 5.63% respectively, meeting the efficacy criterion (not to exceed 7%).
- Growth in exposure plots: Growth was suppressed throughout the exposure period in the 20.0 mg/L plots. 9.09 mg/L plots showed logarithmic growth with some inhibition. 4.13 mg/L plots showed growth similar to the control plots. 1.88 and 0.854 mg/L plots showed growth similar to the control plots.
- Unusual cell shape: In the 20.0 mg/L zone, most cells were swollen, and in the 9.09 mg/L zone, slightly swollen cells were observed. In the other concentrations, the results were similar to those of the control group.
- Colour differences: At the start of exposure, the solution was clear and colourless. At the end of the exposure, the condition of the test solution in the 20.0 mg/L group did not change, and it was slightly pale green in the 9.09 mg/L group and green in the 4.13-0.854 mg/L group due to cell growth. In the control group, the water was clear and colourless at the beginning of the exposure and green at the end of the exposure due to cell growth.

Results with reference substance (positive control):

- Results with reference substance valid? yes
The EbC50 (0-72h) and ErC50 (0-3d) of potassium dichromate (reagent grade, Wako Pure Chemical Industries) of the test organisms were 0.524 and 1.83 mg/L, respectively. The mean ± standard deviation of background data: EbC50 (0-72h) and ErC50 (0-3d) were 0.408 ± 0.079 and 1.12 ± 0.44 mg/L, respectively, n = 10 (including data from 11 April to 14 April 2005, ErC50 (0-3d) includes extrapolated data).

Reported statistics and error estimates:

Method of calculating EC50
The percentage of inhibition for each concentration was plotted on a log-log graph, and a linear regression analysis (least squares method) was carried out using the points of linearity to calculate the EC50 (with 95% confidence limits where possible) from the point of intersection with 50% inhibition. The EC50 (95% confidence limit where possible) was calculated from the point of intersection with 50% inhibition, ErC50 (0-3d) for growth rate, EbC50 (0-72h) for area under the growth curve and EyC50 for yield.

Calculation of the maximum no-effect concentration (NOEC)
For each growth indicator, the Bartlett's test of equal variances was carried out and the presence or absence of significant differences between each concentration group and the control group was determined by one-way analysis of variance and Scheffé's multiple comparison method. However, concentrations higher than the EC50 for each indicator were not used for the significance tests. In addition to the results of these tests of significance, the overall results of the study were taken into account to assess the NOEC.

Validity criteria fulfilled:

yes

Conclusions:

A 72 h-NOEC of 1.88 mg/L based on growth rate and yield and an ErC50 (72h) of 13.5 mg/L was obtained.

Executive summary:

The toxicity of thymol to the aquatic algae Pseudokirchneriella subcapitata was determined in a test conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test) and under 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.

Three test flasks per concentration and six test flasks per control with an initial cell concentration of 10E4 cells/mL each were exposed under static conditions for 72 hours to concentrations of the test substance of nominal 20.0, 9.09, 4.13, 1.88, 0.854 mg/L and control. A 72 h-NOEC of 1.88 mg/L based on growth rate (rounded to 1.9 mg/L) and yield and an ErC50 (72h) of 13.5 mg/L were obtained.

Description of key information

OECD Guideline 201 (Alga, Growth Inhibition Test), GLP; Pseudokirchneriella subcapitata, static, 72 hours, nominal concentrations: 20.0, 9.09, 4.13, 1.88, 0.854 mg/L and control; result: a 72 h-NOEC of 1.88 mg/L (rounded to 1.9 mg/L) based on growth rate and yield and a 72 h-EC50 (growth rate) of 13.5 mg/L were obtained. 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

EC50 for freshwater algae:

13.5 mg/L

EC10 or NOEC for freshwater algae:

1.9 mg/L