[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

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:

17 - 21 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

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: 0.768, 1.92, 4.8, 12.0, 30.0 mg/L (nominal)
- Sampling method: separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates
- Sample storage conditions before analysis: original samples for analysis of menthyl methyl ether were stored at 6 ± 2 °C prior to preparation

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the solutions were stirred with approximately 1100 rpm for 24 hours at room temperature. Appropriate volumes of the test item were pipetted directly into measuring flasks, containing the respective volume of the dilution water. After dispersion treatment the prepared test solutions were transferred to the test vessels which were tightly closed, without headspace

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: SAG 61.81
- Age of inoculum (at test initiation): a 4 days old pre-culture, prepared in dilution water, was used as inoculum
- Method of cultivation: stocks prepared on nutrient medium Z-Agar according to Lüttge et al., 1994 [Lüttge, U, Schnepf, E., Läuchli, A., Nagata, T. (editors, 1994): Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), Thieme- Verlag]. Light intensity was 35-70 µE x m^-2 x s^-1 for 24 hours per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca + Mg/L

Test temperature:

21.5 - 22.5 °C

pH:

initial: 7.91 - 8.16
end of test: 8.32-9.34

Nominal and measured concentrations:

nominal test item concentrations [in mg/L]: 0.768, 1.92, 4.80, 12.0, 30.0
measured concentrations [in mg/L]: 0.612, 1.55, 3.97, 7.95, 17.4 (geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile headspace flasks, with aluminium tops with PTFE seals
- Type: closed
- Headspace, fill volume: no headspace, 119 mL volume
- Initial cells density: nominal: approx. 5 x 10E+03 - 10E+04 cells/mL, current: 6153 cells/mL
- Control end cells density: yes, 760705 cells/mL
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: yes, nutrient medium Z according to Lüttge et al. (1994)

OTHER TEST CONDITIONS
- Photoperiod: 24 h/day light
- Light intensity and quality: 60 - 120 µE x m^-2 x s^-1, fluorescent tubes, cool daylight, homogeneity within ± 15% over incubation area

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorometer, microscope
- Chlorophyll measurement: daily Chlorophyll a fluorescence measurement (excitation at 436 nm, emission at 685 nm)
- Other: microscopic evaluation of the cells at the start and the end of the exposure time (shape, colour, chloroplast morphology, flocculation, agglutination)

TEST CONCENTRATIONS
- Range finding study: non-GLP, closed system without headspace
- Test concentrations: nominal: 1, 10, 100 mg/L, geom. mean measured: 0.469, 7.03, 21.7 mg/L
- Results used to determine the conditions for the definitive study: % growth rate inhibition 4, 23, 100 respective to the geom. mean conentrations 0.469, 7.03, 21.7 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Conf. Interval 9.10 - 10.03 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Conf. Interval 4.04 - 4.92 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Conf. Interval 1.71 - 2.69 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% Conf. Interval 2.79 - 3.65 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.612 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.612 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

Details on results:

- Exponential growth in the control: yes, >= 16-fold corresponding to a specific growth rate of 0.92 per day
- Observation of abnormalities: no
- Unusual cell shape: no

Results with reference substance (positive control):

- Results with reference substance valid: yes
- ErC50: with headspace 0.613 (95% confidence interval 0.589 - 0.638), without headspace 0.774 (95% confidence interval 0.711 - 1.08)

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Table 1: Evaluation of growth rate after 72 hours

Geometric mean of measured test item concentration [mg/L]	Replicate No.	Growth rate [d^-1]	Inhibition of growth rate [%]
17.4	1	<LOQ	100
	2	<LOQ	100
	3	<LOQ	100
	mean	n.a.	100
7.95	1	0.97	39
	2	0.97	39
	3	1.04	35
	mean	1.00	38
3.97	1	1.32	18
	2	1.24	22
	3	1.26	21
	mean	1.28	20
1.55	1	1.53	5
	2	1.53	5
	3	1.51	6
	mean	1.52	5
0.612	1	1.51	6
	2	1.52	5
	3	1.52	5
	mean	1.51	6
control	1	1.64
	2	1.60
	3	1.63
	4	1.56
	5	1.58
	6	1.62
	mean	1.60

Table 2: measured concentrations and percentage of the nominal concentrations of menthyl methyl ether in fresh medium at the beginning of the test and after 72 hours

Sampling timepoint	0 h		72 h
Nominal test item concentration [mg/L]	meas. conc. [mg/L]	[%]	meas. conc. [mg/L]	[%]	Geometric mean measured concentration test item [mg/L]
30	18.40	61	16.40	55	17.40
12	8.19	68	7.72	64	7.95
4.08	3.79	79	4.17	87	3.97
1.92	1.51	79	1.59	83	1.55
0.768	0.607	79	0.618	80	0.612
control	<LOQ		<LOQ		<LOQ

Validity criteria fulfilled:

yes

Conclusions:

The substance was found to inhibit the growth rate of the freshwater green alga Pseudokirchneriella subcapitata after 72 h of exposure. The test was conducted according to the OECD guideline 201 (GLP). ErC10- and ErC50-value were found to be 2.22 mg/L and 9.62 mg/L, respectively.

Description of key information

ErC50 (72h) Pseudokirchneriella subcapitata = 9.62 mg/L (geometric mean measured, OECD 201)

ErC10 (72h) Pseudokirchneriella subcapitata = 2.22 mg/L (geometric mean measured, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

9.62 mg/L

EC10 or NOEC for freshwater algae:

2.22 mg/L

Additional information

One study is available, in which the toxicity to freshwater algae was investigated according to GLP and OECD guideline 201. In a static test system, the test organism Pseudokirchneriella subcapitata was exposed to five nominal concentration of 0.768, 1.92, 4.8, 12.0 and 30.0 mg/L corresponding to geometric mean measured concentrations of 0.612, 1.55, 3.97, 7.95 and 17.4 mg/L. The concentrations of the test item in all test concentrations and the control were analytically verified. The measured concentrations in the fresh media (0 h) were in the range of 61 to 79 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 55 to 87 % of the nominal values of all concentrations. Therefore, all effect values given are based on the geometric mean measured test item concentrations. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 9.62 (9.10 - 10.3) mg/L and 3.25 (2.79 - 3.65) mg/L, respectively. The EC10 values were 2.22 (1.71 - 2.69) mg/L for growth rate and <0.612 mg/L for yield.