L-menthyl acetate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 25, 2012 - Feb 25, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: one undilued filtrate with the loeading rate of 100 mg/L and five dilutions of this filtrate
- Sampling method: For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (with and without algae). At the same sampling times, duplicate samples were also taken from the control. For the aged samples taken at test end, additional flasks containing the test medium with and without algae were incubated for each treatment under the test conditions.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 232.81 mg of the test item in 2320 mL of test water. This preparation was supported by intense stirring on a magnetic stirrer over 3 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.
- Eluate: not applicable
- Differential loading: After the 3-hour stirring period, the dispersion of the test item was left to settle for 1 hour. Thereafter the middle phase of the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm) after preconditioning of the filter with about 200 mL of the dispersion. The negative pressure of the filtration unit was reduced as far as possible to avoid losses of volatile components of the test item during filtration. The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was used in a series of dilution with test water. The test media were prepared just before the start of the test (= start of exposure).
- Controls: water control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up three days before the start of the exposure.
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported, NaHCO3 concentration was increased in order to provide an additional carbon source

Test temperature:

22 °C

pH:

test start: 7.7-7.8
test end: 7.7-8.0
To keep the pH of the test media as constant as possible, 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to the test water.

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

dilutions 1:320, 1:100, 1:32, 1:10, 1:3.2 from the undiluted filtrate of 100 mg test item/L
initially measured: 0.0368, 0.127, 0.35, 1.27, 4.12 and 13.2 mg test item/L
measured after 72 hours: < LOQ, < LOQ, < LOQ, < LOQ, < LOQ, and 12.5 mg test item/L
measured after 72 hours: 0.0596, 0.191, 0.58, 1.955, 4.999 and 13.260 mg test item + Menthol rac./L
geometric mean: 0.047, 0.16, 0.45, 1.6, 4.5 and 13 mg test item + Menthol rac./L

Details on test conditions:

TEST SYSTEM
- Test vessel: since the test item was determined to be volatile, glass stoppered 50 mL Erlenmeyer flasks were used (closed system).
- Type: closed
- Material, size, headspace, fill volume: glass, 60 mL, 0 mL, 60 mL
- Aeration: closed system without aeration in order to minimize losses of the volatile test item
- Initial cells density: 5000 cells/mL
- Control end cells density: 888000 cells/mL (based on the initial cell densityof 5000 algal cells/mL corresponding to 0.50 x 103 relative fluorescence units and the final amount of 88.8 relative fluorescence units)
- No. of organisms per vessel: 5000 cells/mL at test start
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals:
NaHCO3: 250 mg/L
K2HPO4: 1.044 mg/L
MgSO4 × 7 H2O: 14.6 mg/L
MgCl2 × 6 H2O: 12.16 mg/L
CaCl2 × 2 H2O: 4.41 mg/L
NaNO3: 25.5 mg/L
H3BO3: 186.0 μg/L
MnCl2 × 4 H2O: 415.0 μg/L
ZnCl2: 3.27 μg/L
CoCl2 × 6 H2O: 1.43 μg/L
CuCl2 × 2 H2O: 0.012 μg/L
Na2MoO4 × 2 H2O: 7.26 μg/L
FeCl3 × 6 H2O: 160.0 μg/L
Na2EDTA × 2 H2O: 300.0 μg/L
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Conductivity: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: at test start and end

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: To keep the pH of the test media as constant as possible, 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to the test water.
- Photoperiod: 24 hours
- Light intensity and quality: approximately 5300 Lux (range: 4850 to 5800 Lux, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as recommended by the guideline. Use of fluorescent tubes (Philips TLD 36W/840).
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : algal growth rate and biomass
- Determination of cell concentrations: fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm). The measurements were performed at least in duplicate.
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study and definitive study 1
- Test concentrations:
range-finding test: dilutions 1:10 and 1:100 and undiluted filtrate (loading rate 100 mg/L)
definitive study 1: dilutions 1:320, 1:100, 1:32, 1:10, 1:3.2 and undiluted filtrate (loading rate 10 mg/L)
- Results used to determine the conditions for the definitive study:
range-finding test: inhibition of growth rate 7, 19 and 90 % at dilutions 1:100, 1:10 and undiluted filtrate (loading rate 10 mg/L)
definitive study 1: inhibition of growth rate -0.2, -0.2, 1.2, 2.0, 38 and 33 % at 1:320, 1:100, 1:32, 1:10, 1:3.2 and undiluted filtrate (loading rate 10 mg/L); after 72 hours mean measured test item concentrations < LOQ in all treatments.

Reference substance (positive control):

yes

Remarks:

Latest positive control test performed in September 2012: 72-hour EC50 for the growth rate: 0.93 mg potassium dichromate/L (Harlan Study Number D64298), range of 72-hour EC50 for growth rate from 2000 to 2012: 0.71-1.7 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 2.3-3.2 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 0.77-1.3 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 0.42-0.71 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI: 0.59-0.86 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI: 0.25-0.42 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI: 0.15-0.30 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: test item + Menthol rac.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: not reported
- Aggregation of algal cells: no
- Other: no
- Any stimulation of growth found in any treatment: slight increase of growth rate at 0.047 and 0.16 mg/L after 24 hours compared to the control, no increase after 72 hours.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid: yes
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in September 2012 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.93 mg/L (Harlan Study Number D64298), range of the 72-hour EC50 for the growth rate from 2000 to 2012: 0.71-1.7 mg/L).

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95 % confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression [Davis, 1971; Finney, 1971]. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test (one-sided smaller, α = 0.05) [Williams, 1971; Williams, 1972] or Welch t-test (one-sided smaller, α = 0.05) [Sachs, 1984] where appropriate.

Table 1 Average Growth Rates (µ) and inhibition of µ (Ir)

Treatment / Dilution	Mean measured concentration	Average growth rate µ (day^-1) and inhibition of µ (Ir)
		0-24 h		0-48 h		0-72 h
	[mg/L]	µ	Ir[%]	µ	Ir[%]	µ	Ir[%]
Control	---	1.72	0.0	1.74	0.0	1.73	0.0
1:320	0.047	1.73	-1.0	1.75	-0.1	1.71	1.1
1:100	0.16	1.75	-1.9	1.74	0.1	1.70	1.7
1:32	0.45	1.71	0.3	1.69*	3.2	1.63*	5.9
1:10	1.6	1.52*	11.7	1.30*	25.7	1.10*	36.2
1:3.2	4.5	1.18*	31.2	0.89*	48.9	0.71*	58.9
Undiluted filtrate (loading rate 100 mg/L)	13	0.52*	69.5	0.24*	86.3	-0.01*	100.7

*: mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, alpha = 0.05)

Table 2 Yield (Y) and inhibition of Y (Iy)

Treatment / Dilution	Mean measured concentration	Yield Y (x 10^3) and inhibition of Y (Iy)
		0-24 h		0-48 h		0-72 h
	[mg/L]	Y	Iy[%]	Y	Iy[%]	Y	Iy[%]
Control	---	2.3	0.0	15.7	0.0	88.3	0.0
1:320	0.047	2.3	-2.2	15.8	-0.3	83.3	5.7
1:100	0.16	2.4	-4.2	15.7	0.3	81.7	7.5
1:32	0.45	2.3	0.7	14.0*	10.9	65.0#	26.4
1:10	1.6	1.8*	22.0	6.1*	61.0	13.1#	85.2
1:3.2	4.5	1.1*	50.4	2.5*	84.4	3.7#	95.8
Undiluted filtrate (loading rate 100 mg/L)	13	0.3*	84.9	0.3*	98.1	0.0#	100.0

*: mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, alpha = 0.05)

#: mean value statistically significantly lower than in the control (according to Welch t-test, one-sided smaller, alpha = 0.05)

Table 3 Section-by-Section Growth Rates and inhibition of the growth rates (Ir)

Treatment / Dilution	Mean measured concentration	Section-by-section growth rates (day^-1) and inhibition of the growth rates (Ir)
		0-24 h		24-48 h		48-72 h
	[mg/L]	µ	Ir[%]	µ	Ir[%]	µ	Ir[%]
Control	---	1.72	0.0	1.77	0.0	1.70	0.0
1:320	0.047	1.73	-1.0	1.76	0.8	1.64	3.7
1:100	0.16	1.75	-1.9	1.73	2.0	1.61	5.0
1:32	0.45	1.71	0.3	1.66	6.0	1.50	11.4
1:10	1.6	1.52	11.7	1.08	39.3	0.72	57.9
1:3.2	4.5	1.18	31.2	0.60	66.1	0.35	79.5
Undiluted filtrate (loading rate 100 mg/L)	13	0.52	69.5	-0.04	102.5	-0.52	130.4

Table 4 Biomass of Algae

Treatment / dilution	Mean measured concentration	Rep. no.	Biomass of algae*
	[mg/L]		24 hours	48 hours	72 hours
Control	---	1	2.8	16.4	88.1
		2	2.5	16.9	100.2
		3	2.7	16.7	83.4
		4	2.9	15.9	84.9
		5	2.9	14.7	91.6
		6	2.9	16.9	84.7
		Mean	2.8	16.2	88.8
		SD	0.1	0.9	6.3
0.263889	0.047	1	2.7	15.7	77.5
		2	3	16.1	94
		3	2.7	17	79.9
		Mean	2.8	16.3	83.8
		SD	0.2	0.7	8.9
0.111111	0.16	1	3	15.7	78.1
		2	2.7	16.3	99.3
		3	3	16.6	69.1
		Mean	2.9	16.2	82.2
		SD	0.2	0.4	15.5
01:32	0.45	1	2.7	13.9	65.1
		2	2.6	14.5	72.4
		3	2.9	15.2	59.2
		Mean	2.7	14.5	65.5
		SD	0.1	0.7	6.6
01:10	1.6	1	2.3	6.7	12.5
		2	2.4	6.3	14.9
		3	2.1	6.9	13.4
		Mean	2.3	6.6	13.6
		SD	0.2	0.3	1.2
1:3.2	4.5	1	1.8	3.1	3.6
		2	1.6	2.9	4.5
		3	1.5	2.8	4.5
		Mean	1.6	2.9	4.2
		SD	0.1	0.1	0.5
Undiluted filtrate (loading rate	13	1	0.9	0.7	0.5
100 mg/L)		2	0.8	0.8	0.5
		3	0.8	0.9	0.4
		Mean	0.8	0.8	0.5
		SD	0	0.1	0

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 103). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.50 x 103 relative fluorescence units.

Validity criteria fulfilled:

yes

Conclusions:

EC50 for 72-hour for growth rate: 2.7 mg test item + Menthol rac./L (95 % confidence interval: 2.3-3.2 mg/L)
EC10 for 72-hour for growth rate: 0.61 mg test item + Menthol rac./L (95 % confidence interval: 0.42-0.81 mg/L)

Executive summary:

In a reliable, conclusive and valid study, the influence of the test item Menthyl Acetate racemic on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated under 72 hour staticconditions according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3.

Due to the low solubility of the test item in test water, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 3 hours. This dispersion of the test item was left to settle for 1 hour. Thereafter the middle phase of the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm) after preconditioning of the filter with about 200 mL of the dispersion. The undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:3.2, 1:10, 1:32, 1:100 and 1:320 were used as test media. Additionally, a control was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

At the start of the test, the analytically determined concentrations of Menthyl Acetate racemic in the test media (dilutions 1:320, 1:100, 1:32, 1:10, 1:3.2 and the undiluted filtrate) were 0.0368, 0.127, 0.35, 1.27, 4.12 and 13.2 mg/L, respectively. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, the measured test item concentrations in the test media with algae (dilutions 1:320 to 1:3.2) were below the Limit of Quantification (LOQ=0.0218 mg/L). In the undiluted filtrate with algae, 12.5 mg/L was found.

Additionally, the test media were incubated over the test duration, but without algae. The measured test item concentrations in the test media without algae (dilutions 1:320 to 1:3.2) were below the LOQ. In the undiluted filtrate without algae, 9.2 mg/L was found. Therefore the losses in the test media with algae during the test period were not considered to be due to adsorption of the test item onto the algae.

Since most of the test item was degraded at the end of the test, the analytical measurements were also based on Menthol rac. (CAS 89-78-1), a degradation product of the test item. At the end of the test, the total determined concentrations of Menthol rac. (expressed as test item) plus test item were 0.0596, 0.191, 0.58, 1.955, 4.999 and 13.26 mg/L, respectively. The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start (test item concentration) and end (Menthol rac. concentrations expressed as test item + test item) of the test.

The 72-hour NOEC based on the growth rate and biomass were determined to be 0.16 mg/L, since up to and including this mean measured concentration the growth rate of the algae after 72°hours was not significantly lower than in the control. The EC50 -values were as follows:

EC50 for 72-hour for growth rate: 2.7 mg test item + Menthol rac./L (95 % confidence interval: 2.3-3.2 mg/L)

EC10 for 72-hour for growth rate: 0.61 mg test item + Menthol rac./L (95 % confidence interval: 0.42 -0.81 mg/L)

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Dec 10, 2002 - Dec 13, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Schedule of sampling:
Stock solution at 0 hours
Control at 0 and 72 hours
Test concentration at 0 and 72 hours

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Before the beginning of the study, the test substance was pulverized. A stock solution was prepared to give the desired series of test concentrations. To achieve this 124.9 mg of the test substance were added to 1 litre of dilution water and treated for 1 h in an ultrasonic bath and afterwards stirred for 24 h on a magnetic stirrer.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Non-axenic strain
- Source (laboratory, culture collection): "The collection of Algal Cultures" of the Institute of Plant Physiology at the University of Göttingen, Germany.
- Method of cultivation: Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions (23 +/- 2°C) at a light intensity in the range 60-120 μE/m2/s (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KÜHN (1977)) is renewed once a week. Cell density measurements are made using a microcell counter.

ACCLIMATION
- Acclimation period: Pre-cultures are set up three days before the start of a test.
- Culturing media and conditions (same as test or not): Same as test culture method (see above)

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21°C to 25°C +/- 2°C

pH:

At 0 hour: pH=8.2 to 8.5
At 72 hours: pH=8.6 to 10.4

Nominal and measured concentrations:

Nominal concentrations: 5.0, 10, 20, and 40 mg/l administered from stock solutions.
Measured concentrations (at 0 hour - at 72 hours): 4.6-4.5, 10-9.3, 19.9-17.6 and 41-36.7 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 ml Erlenmeyer flasks with stoppers
- Initial cells density: 10^4 cells/ml
- Control end cells density: 318889 cell/ml
- No. of vessels per concentration (replicates): 3 replicates per concentration
- No. of vessels per control (replicates): 6 replicates per concentration

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: continuous uniform illumination
- Light intensity and quality: light between 400-700 nm, light intensity in the range 60-120 μE/m2/s (equivalent to 4000 to 8000 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microcell counter or alternatively by means of a microscopic counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2
- Test concentrations: see above

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: reduction of growth

Remarks on result:

other: 95% confidence limits : 16-24 mg/l

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits : 21.4-21.5 mg/l

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.65 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: reduction of growth

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.65 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.:

18.75 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: reduction of growth

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

18.75 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

yes

Conclusions:

In the test using the freshwater algae Desmodesmus subspicatus an ErC50 (based on growth rate) of 21.4 mg/l was determined, whereas the No effect concentration (NOEC) was determined to be 9.65 mg/l.

Description of key information

ErC50 (72 h) = 2.7 mg/L (measured, OECD 201); read-across
ErC10 (72 h) = 0.61 mg/L (measured, OECD 201); read-across

Key value for chemical safety assessment

EC50 for freshwater algae:

2.7 mg/L

EC10 or NOEC for freshwater algae:

0.61 mg/L

Additional information

Since no data for the target substance (L-menthyl acetate, CAS 2623-23-6) are available, the aquatic toxicity is assessed on the basis of data from one structurally similar substance (menthyl acetate, CAS 89-48-5) and a possible product of hydrolysis (L-menthol,CAS 2216-51-5). A detailed justification of the analogue approach is provided in the overall summary and in the technical dossier in IUCLID Section 13.

The key study with the source substance menthyl acetate was performed according to OECD 201 (GLP). Pseudokirchneriella subcapitata was exposed to a serial dilution (1:320, 1:100, 1:32, 1:10, 1:3.2) of a stock solution of 100 mg/L. The actual exposure concentrations were measured using a suitable analytical method measuring the test item and the hydrolysis product. It was shown that the test item was degraded after 72 h. Compared to the initially measured concentrations the measured concentrations dropped below the LOQ at all concentrations (except for the highest test concentration). Additionally, the test media were incubated over the test duration, but without algae. The measured test item concentrations in the test media without algae (dilutions 1:320 to 1:3.2) were below the LOQ. In the undiluted filtrate without algae, 9.2 mg/L was found. Therefore the losses in the test media with algae during the test period were not considered to be due to adsorption of the test item onto the algae.

At the same time the hydrolysis product Menthol rac. (CAS 89 -78 -1) was identified to be present in the test solutions. Since most of the test item was degraded at the end of the test, the analytical measurements were also based on Menthol rac. (CAS 89-78-1), a degradation product of the test item. At the end of the test, the total determined concentrations of Menthol rac. (expressed as test item) plus test item were 0.0596, 0.191, 0.58, 1.955, 4.999 and 13.26 mg/L, respectively. The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start (test item concentration) and end (Menthol rac. concentrations expressed as test item + test item) of the test.

The biological results (based on geometric mean measured concentrations) of the study were as follows: EC50 growth rate is 2.7 mg/L. EC10 growth rate is 0.61 mg/L. NOEC growth rate is 0.16 mg/L. In a supporting study the effect of the substance on different species of saltwater algae was tested and algal toxicity was observed at a test level of 100 ppm, which supports the finding of the key study.

The supporting study with L-menthol, performed according to EU Method C.3 (GLP), resulted in a higher ErC50 (72 h) of 21.4 mg/L (measured) and a NOErC (72 h) of 9.65 mg/L.

Based on the results from one structurally related source substance and the expected abiotic hydrolysis product (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) it can be concluded that L-menthyl acetate has toxic effects on freshwater algae.