Sodium mentholate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-02-15 - 2021-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

corrected version as of July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document on aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals No. 23 (2019)

Version / remarks:

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals", 2nd Ed., February 08, 2019

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 7.5, 2.4, 0.75, 0.24 and 0.075 mg/L and control
- Sampling method: The samples were taken from the biological phase of the study. Quadruplicate samples from the freshly prepared test media (without algae) of all test concentrations and from the control were taken at the start of the test. Quadruplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test by pouring together the contents of the test beakers of each treatment.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ -20 °C), protected from light, until liquid-liquid extraction and analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest test concentration of nominal 7.5 mg test item/L was prepared by dissolving 7.4 mg test item into 987 mL test water by intense stirring for 90 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
- Controls: negative control containing test water (OECD Medium) without addition of test item
- Test concentration separation factor: 3.16
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations. All test media were clear and colourless throughout the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): original supplier: „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Water temperature was 22.0 to 22.5 °C
The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

pH:

control: 8.1 at test start; 9.6 at test end
test item treatments: 7.9 - 8.1 at test start; 8.7 - 9.6 at test end

Nominal and measured concentrations:

Nominal: 7.5, 2.4, 0.75, 0.24 and 0.075 mg test item/L and a control
Measured: 5.42, 1.71, 0.556, 0.179 and 0.0646 mg test item/L and a control

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of at least 50 mL volume
- Type: closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation
- Headspace, fill volume: as much test medium as possible (i.e. the remaining head space was reduced to a technical possible minimum of some millilitres)
- Initial cells density: nominal 5000 algal cells per mL
- Control end cells density: 109.276 [1000/mL]
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Additional replicates: One replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as the other replicates.

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared 4 days before test start to allow pH to stabilise.
- Hardness: 0.24 mmol/L (=24 mg/L) as CaCO3
- pH before test start: 8.1 +/- 0.1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity: 5473 lux (range: 5070 to 5810 lux)
- Test procedure: The test units were continuously stirred by magnetic stirrers. The flasks were incubated in a water bath, were placed in a random order and were repositioned each day to minimize differences in test conditions.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [spectrophotometer]
The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test
- Signs of Phytotoxicity evaluated by Microscopic Observations: To check for any effect of the test item on the morphology of the algal cells, at least one sample from all test concentrations and the control was taken after the test period of 72 hours. The shape of the treated algal cells compared to the control was examined microscopically.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
- Test concentrations: not specified
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

reference item potassium dichromate is tested at least twice a year, last performance September 2020

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.86 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.17 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.71 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.556 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.08 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.671 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.71 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.556 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: The microscopic examination of the shape of the algal cells after 72hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 7.5 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
- Any stimulation of growth found in any treatment: Yes, but the stimulation of growth was not dose related and only slight within the considered natural biological variation.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.402 mg/L for yield, 0.878 mg/L for growth rate, 0.449 mg/L for biomass

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by a three parametric normal cumulative distribution function (CDF).
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Bonferroni-Welch t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD 201 taking into account the OECD guidance document no. 23 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards freshwater algae.
Exponentially growing cultures of the unicellular green algal species Pseudokirchneriella subcapitata were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part by GC-MS. Based on the arithmetic mean measured concentrations of the test item the 72-hour EyC50 was calculated to be 1.08 mg test item/L and the 72-hour ErC50 value was calculated to be 1.86 mg test item/L. The 72-hour NOEyC was determined to be 0.556 mg test item/L and the associated 72-hour LOEyC was 1.71 mg test item/L based on arithmetic mean measured test concentrations. The 72-hour NOErC was determined to be 0.556 mg test item/L and the associated 72-hour LOErC was 1.71 mg test item/L, both values also based on arithmetic mean measured test concentrations.

Executive summary:

The study was conducted under GLP according to OECD 201 and taking into account the OECD guidance document no. 23. The purpose of this test was to determine the inhibitory effect of the registered substance on the growth of the freshwater green algae Pseudokirchneriella subcapitata.

For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to concentrations nominal 7.5, 2.4, 0.75, 0.24 and 0.075mg test item/L and a controlunder defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. To avoid loss of test item due to volatilisation, the test vessels were kept closed with a reduced headspace during the exposure period of 72 hours. The test conditions (water temperature, dissolved oxygen content, pH and light) were within the ranges requested by OECD TG 201 and the validity criteria stated in the guideline were met.

The initial concentrations and the maintenance of the exposure concentrations during the test were analytically verified by GC-MS. The quantification of the test item was performed indirectly by analysing the hydrolysis product menthol in the test samples using liquid/liquid extraction followed by measurement via gas chromatography with MS detection. Direct analysis of the test material was not feasible due to its hydrolysis instability (DT50< 5min). Since the test item concentrations were not within ± 20% of the nominal concentrations during the test, the biological results refer to arithmetic mean measured concentrations.

Based on the arithmetic mean measured concentrations of the test item the 72-hour E_yC₅₀was calculated to be 1.08 mg test item/L and the 72-hour E_rC₅₀value was calculated to be 1.86 mg test item/L. The 72-hour NOE_yC was determined to be 0.556 mg test item/L and the associated 72-hour LOE_yC was 1.71 mg test item/L based on arithmetic mean measured test concentrations. The 72-hour NOE_rC was determined to be 0.556 mg test item/L and the associated 72-hour LOE_rC was 1.71 mg test item/L,both values also based on arithmetic mean measured test concentrations.

Description of key information

1) Key_Toxicity to aquatic algae and cyanobacteria: E_rC₅₀(72h) = 1.86 mg/L, E_rC₁₀(72h) = 1.17 mg/L and NOE_rC (72h) = 0.556 mg/L based on arithmetic mean measured concentrations for Pseudokirchneriella subcapitata (static, freshwater, OECD 201, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

1.86 mg/L

EC10 or NOEC for freshwater algae:

1.17 mg/L

Additional information

The study was conducted under GLP according to OECD 201 and taking into account the OECD guidance document no. 23. The purpose of this test was to determine the inhibitory effect of the registered substance on the growth of the freshwater green algae Pseudokirchneriella subcapitata.

For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to concentrations nominal 7.5, 2.4, 0.75, 0.24 and 0.075mg test item/L and a controlunder defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. To avoid loss of test item due to volatilisation, the test vessels were kept closed with a reduced headspace during the exposure period of 72 hours. The test conditions (water temperature, dissolved oxygen content, pH and light) were within the ranges requested by OECD TG 201 and the validity criteria stated in the guideline were met.

The initial concentrations and the maintenance of the exposure concentrations during the test were analytically verified by GC-MS. The quantification of the test item was performed indirectly by analysing the hydrolysis product menthol in the test samples using liquid/liquid extraction followed by measurement via gas chromatography with MS detection. Direct analysis of the test material was not feasible due to its hydrolysis instability (DT50< 5min). Since the test item concentrations were not within ± 20% of the nominal concentrations during the test, the biological results refer to arithmetic mean measured concentrations.

Based on the arithmetic mean measured concentrations of the test item the 72-hour E_yC₅₀was calculated to be 1.08 mg test item/L and the 72-hour E_rC₅₀value was calculated to be 1.86 mg test item/L. The 72-hour NOE_yC was determined to be 0.556 mg test item/L and the associated 72-hour LOE_yC was 1.71 mg test item/L based on arithmetic mean measured test concentrations. The 72-hour NOE_rC was determined to be 0.556 mg test item/L and the associated 72-hour LOE_rC was 1.71 mg test item/L,both values also based on arithmetic mean measured test concentrations.