Essential oil of Mentha Spicata L., Erospicata (Labiatae) obtained from aerial parts by steam 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:

18 - 21 Oct 2016

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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 0.625, 1.25, 2.5, 5.0, and 10.0 mg/L (nominal test item loading)
- Sampling method: samples for test item analysis were taken at 0, 48, and 72 h
- Sample storage conditions before analysis: all samples were stored at 6 ± 2°C until sample preparation and, if necessary, at RT (on an autosampler) until the start of the analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Five water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 0.625, 1.25, 2.5, 5.0, and 10 mg/L. An appropriate amount of the test item was directly pipetted into a glass bottle with an appropriate amount of dilution water for each loading. This dispersion was shaken for 23 h at 20 rpm and room temperature. After a separation phase of 1 h, the WAF was removed by siphoning (from the approximate centre of the flask) and used in the test.
- Differential loading: yes
- Controls: dilution water without test item

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Age of inoculum at test initiation: 4 d

CULTIVATION
- Method: Fresh stocks are prepared every month on Z-agar. Light intentsity amounted to 2567 - 5130 lux corresponding to 35 - 70 µE * m-² * s-1 for 24 h per day.
- Culture medium: nutrient medium Z, according to Lüttge et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L (dilution water)

Test temperature:

Mean = 21.5 °C, Min. = 21.0 °C, Max = 22.0 °C

pH:

0 h: 8.13 (control), 8.08 - 8.11 (treatments)
72 h: 9.35 (control), 8.57 - 9.29 (treatments)

Nominal and measured concentrations:

control, 0.625, 1.25, 2.50, 5.00, and 10.0 mg/L (nominal test item loading)
control, 0.750, 1.30, 2.59, 5.05, and 10.2 mg/L (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile headspace flasks
- Type: closed, with aluminium tops with PTFE seals
- Material, size, headspace, fill volume: Flask volume: 59 mL; Test volume: 59 mL; Headspace: 0 mL
- Initial cells density: 6305 cells/mL
- Control end cells density: 518869 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, nutrient medium Z, according to Lüttge et al. (1994)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guidelines
- pH: 8.1 ± 0.2
- Culture medium different from test medium: yes, test solutions were prepared in dilution water
- Intervals of water quality measurement: pH values were measured at the beginning and end of the test. The pH-values at the start of exposure were measured in one additional replicate of each test item loading and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item loading and the control. The room temperature (RT) was measured continuously and the light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h light/d
- Light intensity and quality: 5456 lux (mean), CV = 6.65%
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm. The flasks were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: chlorophyll-a fluorescence, measured by fluorometer (Microplate Reader Chameleon V (Hidex) with Software Micro Win 4.41 (Mikrotec Laborsysteme GmbH), at the wavelengths 436 nm (excitation) and 685 nm (emisison).
- Chlorophyll measurement: at 0, 24, 48, and 72 h
- Other: microscopic evaluation of the cells (shape, color, morphology, behaviour) at the start and the end of incubation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes, non-GLP
- Test concentrations of range finding study: 1.0, 10.0, and 100 mg/L (nominal loading level of the water accommodated fraction)
- Results used to determine the conditions for the definitive study: For the loading level of 100 mg/L a Tyndale effect was observed and it was visually turbid throughout the exposure period. The other loading levels showed no Tyndale effect and were visually clear throughout the exposure period. 100% growth rate inhibition and yield inhibition were observed after 72 h at a nominal loading level of 10.0 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

8.29 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. (WAF)

Basis for effect:

growth rate

Remarks on result:

other: 95% CI

Remarks:

5.44 - 9.97 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

5.19 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. (WAF)

Basis for effect:

growth rate

Remarks on result:

other: 95% CI

Remarks:

2.37 - 9.94 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.625 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. (WAF)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.742 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. (WAF)

Basis for effect:

other: yield

Remarks on result:

other: 95% CI

Remarks:

0.646 - 0.87 mg/L

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

6.18 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. (WAF)

Basis for effect:

other: yield

Remarks on result:

other: 95%CI

Remarks:

5.15 - 6.99 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50 (72 h): 0.435 mg/L (with headspace), 0.811 mg/L (without headspace)

Reported statistics and error estimates:

EL10, EL20, and EL50 values with confidence intervals of growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression and third order polynomial regression. The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield. The following statistical tests were conducted: Monotonicity was done by trend analysis by contrasts (significance levels 0.05). Shapiro-Wilk´s test on normal distribution was done with a significance level of 0.01. Levene´s test on variance homogeneity was done wtih a significance level 0.01. William´s test on significant differences was done with a significance level 0.05.

The measured concentrations of the test item at the start of the exposure were in the range of 101 -120%. After 48 h, the measured concentrations were in the range of 104 - 122%. At test end, the test item concentrations were in the range of 99 - 122%.

Table 1: Evaluation after 72 h. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item loading [mg/L]	Replicate n°	Growth rate [d-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
10.0	1	n.a.	100	n.a.	100
	2	n.a.	100	n.a.	100
	3	n.a.	100	n.a.	100
	mean	(+)    n.a.	100	(+)    n.a.	100
5.0	1	1.33	9	338697	34
	2	1.34	9	347492	32
	3	1.34	9	343292	33
	mean	(+)   1.34	9	(+) 343160	33
2.5	1	1.30	11	307237	40
	2	1.32	10	325984	36
	3	1.31	11	311945	39
	mean	(+)   1.31	11	(+) 315055	39
1.25	1	1.39	5	407368	21
	2	1.41	4	428511	16
	3	1.34	9	344476	33
	mean	(+)   1.38	6	(+) 393452	23
0.625	1	1.45	2	477646	7
	2	1.51	-3	582456	-14
	3	1.46	0	503552	2
	mean	(-)    1.47	0	(-) 521218	-2
control	1	1.47		520128
	2	1.48		530220
	3	1.50		563231
	4	1.48		528247
	5	1.42		433557
	6	1.46		500000
	mean	1.47		512564

Negative inhibition = growth stimulation.

n.a. = data not determinable.

Table 2. NOEC, LOEC, ECx-values and 95% Confidence Intervals of test item (0 – 72 h) based on nominal test item loadings [mg/L]

	Growth Rate Inhibition Nominal test item loading [mg/L]
NOEL	0.625
LOEL	1.25
ErL10	5.19 (2.37 – 9.94)
ErL20	6.53 (5.12 – 9.95)
ErL50	8.29 (5.44 – 9.97)
	Yield Inhibition Nominal test item loading [mg/L]
NOEL	0.625
LOEL	1.25
EyL10	0.742 (0.646 – 0.870)
EyL20	0.893 (0.757 – 1.31)
EyL50	6.18 (5.15 – 6.99)

Validity criteria fulfilled:

yes

Conclusions:

ErL50 (72 h) = 8.29 mg/L (nominal loading level, Pseudokirchneriella subcapitata, OECD 201)
ErL10 (72 h) = 5.19 mg/L (nominal loading level,Pseudokirchneriella subcapitata, OECD 201)

Description of key information

ErL50 (72 h) = 8.29 mg/L (nominal loading level, Pseudokirchneriella subcapitata, OECD 201)

ErL10 (72 h) = 5.19 mg/L (nominal loading level, Pseudokirchneriella subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

8.29 mg/L

EC10 or NOEC for freshwater algae:

5.19 mg/L

Additional information

There is one GLP certified guideline study available, which investigated the effects of the test substance on the growth of freshwater microalgae according to the OECD guideline 201.

In a static test Pseudokirchneriella subcapitata was exposed to five test concentrations in closed bottles without headspace for 72 h under controlled conditions. Since the test substance is a UVCB with partly poorly soluble components, five water accommodated fractions (WAF) with the nominal test item loading levels ranging from 0.625 to 10 mg/L were prepared in dilution water and shaken for 24 h. The concentrations of the test substance in the test solutions were analytically verified by GC-MS and ranged from 99 – 122% of the nominal loading concentration. Therefore, all effect values are based on the nominal test item loading rates.

The EL50 (72 h) for inhibition of growth rate was found to be 8.29 mg/L (95% CI = 5.44 – 9.97) and the EL10 for inhibition of growth rate was 5.19 mg/L (95% CI = 2.37 – 9.94).