Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

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:

8/12/2014 - 22/01/2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study according to international guideline (OECD guideline 201) under GLP. No deviations from guideline reported.

Reason / purpose for cross-reference:

reference to same 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

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

spearmint oil
Mentha cardiaca, ext.
EC 294-809-8
CAS 91770-24-0
Oil of spearmint - Chinese type
Oil of spearmint - Scotch variety

Details on properties of test surrogate or analogue material (migrated information):
Not relevant

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal water accommodated fractions (WAF) of 0.32, 1.0, 3.2, 10, 32 and 100 mg/l
- Sampling method: 50 ml samples were taken at t=0 and t=72 hours. It should be noted that samples were taken from solutions incubated without algae and HEPES buffer.
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Spearmint Essential oil, ex Mentha cardiaca is a clear colourless to yellow liquid and a mixture of components with poor water solubility.

Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging from 0.32 to 100 mg/l. Exact amounts of test substance were added to test medium containing no HEPES buffer and magnetically stirred for 2 days in closed vessels with minimal headspace. Thereafter, all solutions were left to stabilise overnight. Subsequently, the WAFs were collected by siphoning the water phase.

After preparation, volumes of 110 ml were added to each vessel containing 2.2 ml of an algal suspension (providing a cell density of 10^4 cells/ml) and HEPES buffer (0.66 ml) was spiked to each vessel.
Solutions for analysis of TOC concentrations:
• At the start of the test samples were taken from freshly prepared solutions (before preparation of the exposure vessels).
• At the end of the exposure period volumes of 110 ml of were added to each vessel containing no algae. These vessels were not spiked with HEPES buffer.

This was done to prevent that the carbon originating from the buffer will obscure the results.
- Controls: Yes, test medium without test substance or other additives

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not relevant

Hardness:

24 mg/l CaCO3

Test temperature:

22 - 23ºC

pH:

7.1 - 7.7

Dissolved oxygen:

Not relevant

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal: WAFs of control, 0.32, 1.0, 3.2, 10, 32 and 100 mg/l
Measured (t=0h, TOC): -0.2293, 0.01529, -0.03736, 1.883, 6.00, 21.44 and 62.61 mg/l
Measured (t=72h, TOC): -0.6087, -0.498, -0.2505, 1.283, 5.316, 19.22 and 57.43 mg/l
Concentrations below 1.0 mg TOC/l cannot be reliably quantified

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 120 ml, all-glass, containing 110 ml of test solution, closed airtight.
- Aeration: none
- Initial cells density: 1 x 10^4 cells/ml
- Control end cells density: 112.1 x 10^4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, adjusted M2 medium
- Composition of the medium: adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 300 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
HEPES 6 mmol/l
pH 7.1 ± 0.3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis
- Culture medium different from test medium: No
- Intervals of water quality measurement:
- pH: At the beginning and at the end of the test. The pH of the control solutions should preferably not deviate by more than 1.5 units during the test.
- Temperature of medium: Continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps, 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank.
- Other: t the end of the final test microscopic observations were performed at the WAF of 32 mg/l to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 100 mg/l
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 14-27 mg/l

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 23 - 38 mg/l

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 56 - 76 mg/l

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 0.57 - 6.9 mg/l

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% Cl: 1.9 - 12 mg/l

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% Cl: 13 - 49 mg/l

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Any stimulation of growth found in any treatment: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 72hErC50: 1.5 mg/l; 82hEbC50: 0.41 mg/l

Reported statistics and error estimates:

The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments. The average growth rate at each test substance concentration is then compared with the control value and the percentage inhibition.

For determination of the NOELR and the EL50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Additionally, the EL10 and EL20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

Calculation of ELx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding loading rate of the test substance.

The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Growth rates were in the range of the controls at the loading rates ranging from 0.32 to 10 mg/l during the 72-hour test period, whereas the growth rate of algae exposed to WAFs prepared at 32 and 100 mg/l were increasingly reduced. Due to mathematical reasons statistical analysis could not be performed for the data gathered after 72 hours of exposure. Therefore, the NOELR for growth rate inhibition could not be determined. Instead, the EL₁₀was calculated as an equivalent alternative.

 

Inhibition of yield increased with increasing loading rate of Spearmint Essential oil, ex Mentha cardiaca from 3.2 mg/l upwards resulting in 97% inhibition at the highest loading rate tested. Statistically significant inhibition of yield was found at the two highest WAFs. It should be noted that at the two lowest WAFs an inhibition of 26 and 24% was observed. However, it was assumed to be not related to the test substance as the TOC concentrations measured in these WAFs were below the concentrations measured in the higher loading rates

Percentage inhibition of growth rate (total test period) during the final test

Spearmint Essential oil, ex Mentha cardiaca WAF prepared at (mg/l)	Mean	Std. Dev.	n	%Inhibition
Control	1.570	0.0539	6	0.0
0.32	1.472	0.0180	3	6.2
1.0	1.483	0.0278	3	5.5
3.2	1.560	0.0413	3	0.6
10	1.538	0.0275	3	2.0
32	1.244	0.2231	3	20.8
100	0.486	0.0117	3	69.0

Percentage inhibition of growth rate at different time intervals during the final test

Spearmint Essential oil,  ex Mentha cardiaca WAF prepared at (mg/l)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.950	0.0	1.542	0.0	1.217	0.0
0.32	3	2.068	-6.1	1.490	3.4	0.858	29.5
1.0	3	2.129	-9.2	1.394	9.6	0.927	23.8
3.2	3	2.052	-5.2	1.473	4.5	1.157	4.9
10	3	2.160	-10.8	1.282	16.9	1.173	3.6
32	3	2.009	-3.1	0.845	45.2	0.877	27.9
100	3	1.773	9.0	0.115	92.5	-0.430	135.3

Percentage inhibition of yield during the final test

Spearmint Essential oil, ex Mentha cardiaca WAF prepared at (mg/l)	Mean	Std. Dev.	n	%Decrease
Control	111.059	16.3530	6	0.0
0.32	81.852	4.5147	3	26.3*
1.0	84.823	7.3310	3	23.6
3.2	107.456	12.9902	3	3.2
10	100.243	8.4980	3	9.7
32	46.443	24.9677	3	58.2*
100	3.304	0.1495	3	97.0*

* - effect was statistically significant

Individual cell densities (x10⁴cells/ml)

Time	Replicate	Spearmint Essential oil, ex Mentha cardiaca WAF prepared at (mg/l)
		Control	0.32	1.0	3.2	10	32	100
0 h	1 2 3 4 5 6	1.0	1.0	1.0	1.0	1.0	1.0	1.0
	2	1.0	1.0	1.0	1.0	1.0	1.0	1.0
	3	1.0	1.0	1.0	1.0	1.0	1.0	1.0
	4	1.0
	5	1.0
	6	1.0
n:		6	3	3	3	3	3	3
Mean:		1.0	1.0	1.0	1.0	1.0	1.0	1.0
Std.Dev.:		0.0	0.0	0.0	0.0	0.0	0.0	0.0
CV:		0.0	0.0	0.0	0.0	0.0	0.0	0.0
24 h	1 2 3 4 5 6	6.813	8.486	8.581	8.339	8.142	9.319	5.575
	2	8.559	7.472	8.987	7.804	8.705	6.442	5.462
	3	5.912	7.810	7.714	7.241	9.206	6.909	6.712
	4	7.512
	5	6.932
	6	6.706
n:		6	3	3	3	3	3	3
Mean:		7.1	7.9	8.4	7.8	8.7	7.6	5.9
Std.Dev.:		0.9	0.5	0.7	0.5	0.5	1.5	0.7
CV:		12.6	6.5	7.7	7.0	6.1	20.4	11.7
48 h	1 2 3 4 5 6	31.206	35.013	33.870	29.967	27.901	25.170	6.588
	2	31.673	33.684	38.025	37.152	33.487	10.254	7.545
	3	32.135	36.696	30.232	35.125	32.642	20.294	5.811
	4	34.399
	5	36.020
	6	31.961
n:		6	3	3	3	3	3	3
Mean:		32.9	35.1	34.0	34.1	31.3	18.6	6.6
Std.Dev.:		1.9	1.5	3.9	3.7	3.0	7.6	0.9
CV:		5.7	4.3	11.5	10.9	9.6	41.0	13.1
72 h	1 2 3 4 5 6	110.460	81.760	81.551	93.686	94.463	67.339	4.431
	2	122.353	78.984	94.288	118.107	98.489	19.426	4.341
	3	111.626	87.813	81.630	113.574	110.776	55.565	4.139
	4	119.881
	5	126.795
	6	81.236
n:		6	3	3	3	3	3	3
Mean:		112.1	82.9	85.8	108.5	101.2	47.4	4.3
Std.Dev.:		16.4	4.5	7.3	13.0	8.5	25.0	0.1
CV:		14.6	5.4	8.5	12.0	8.4	52.6	3.5

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 112. The test fulfils this validity criterion.

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by a factor > 15, coefficients of variation did not exceed guideline limits

Conclusions:

The acute toxicity (72h-ErL50) of Spearmint oil towards Pseudokirchneriella subcapitata is 65 mg/l.

Executive summary:

The acute toxicity of Spearmint oil towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to nominal WAFs with loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/l and were observed 72 hours. Based on nominal concentrations the 72h-ErL10 and 72h-ErL50 were found to be 21 and 65 mg/l respectively.

Description of key information

The 72h-ErL10 and 72h-ErL50 of Spearmint oil towards Pseudokirchneriella subcapitata are 21 and 65 mg/l respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

65 mg/L

EC10 or NOEC for freshwater algae:

21 mg/L

Additional information

The acute toxicity of Spearmint oil towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to nominal WAFs with loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/l and were observed 72 hours. Based on nominal concentrations the 72h-ErL10 and 72h-ErL50 were found to be 21 and 65 mg/l respectively