4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

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:

From 14 July 2015 to 13 August 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)

Version / remarks:

(2006; Annex 5 corrected 28 July 2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2008, amended in 2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples of 3.0 mL from the approximate centre of the test vessels were taken from all test concentrations at t=0, 24, 48 and 72 h. Samples were stored in a freezer until analysis. At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling, instead, samples were taken from one vessel per concentration.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of Orange Flower Ether tested was a colourless liquid with a purity of 83.8% and not completely soluble in test medium at the concentrations tested. Water Accommodated Fractions (WAFs) were prepared in closed vessels at loading rates ranging from 1.0 to 100 mg/L applying a 2-day period of magnetic stirring followed by a one-hour stabilisation period. The aqueous fractions were collected by siphoning and used as test solutions. The final test solutions were all clear and colourless.

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: in-house laboratory culture

ACCLIMATION
- Stock culture medium: M1
- Pre-culture:
3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) 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.

After preparation, volumes of appr. 120 mL test solution were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. The total fill volume of each vessel was appr. 120 mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L (as CaCO3).

Test temperature:

Temperature was maintained between 22 and 23°C throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.4 - 7.7.

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L (based on range-finding test).
- Measured concentrations: n.d. 0.41, 2.0, 4.3, 12 and 37 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 mL all-glass vessels were used, each containing 120 mL of test preparation for the control and each treatment group. The vessels were air-tight closed and there was minimal headspace to prevent loss of test substance due to volatilisation. The control group was maintained under identical conditions but not exposed to the test material.
- Initial cells density: 1E+04 cells per mL
- No. of vessels per concentration (replicates): 3 + 2 extra replicates of each test concentration for sampling purposes at t=24 h and t =48 h
- No. of vessels per control (replicates): 6
- No. of vessels without algae (replicates): 1 (only samples taken from the vessel with a loading rate of 10 mg/L were analysed)

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture and test medium: adjusted M2 (prepared in accordance with OECD 201 using reverse osmosis purified deionised water (Milli-RO, Millipore), pH adjusted to 8.1 ± 0.2)
- llumination: continuously using TLD-lamps with a light intensity within the range of 62 to 70 µE.m-2.s-1 while constantly shaking.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Samples were taken at t = 0, 24, 48 and 72 hours and the cell densities were determined. 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 =20 mm. Algal medium was used as blank.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the two highest test concentrations to observe for any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 25 - 26 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 11- 12mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Statistically significant inhibition of growth rate was found at geometric mean measured concentrations of 12 mg/L and higher. The NOEC is set at 4.3 mg/L.
- Statistically significant inhibition of yield was observed in all test solutions at geometric mean measured concentrations of 12 mg/L, ranging from 40.2 to 97.8 % inhibition.

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

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-h ErC50: 1.3 mg/L (95% confidence interval ranging from 1.2 to 1.5 mg/L). test performed June 2015.
- Other: The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEC and the EC50, 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).

Calculation of ECx 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 average exposure concentrations of the test substance.

The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Table: Percentage inhibition of growth rate and yield after 72 hours exposure 

Mean measured conc. (mg/L)	Percentage inhibition
	Growth rate	Yield
0 (control)	-	-
0.41	-0.4	-1.6
2.0	-0.7	-3.1
4.3	1.4	7.0
12	10.4*	40.2*
37	72.8*	97.8*

* statistically significant effect

Table: measured test concentrations

Loading rate (mg/L)	Measured concentrations (mg/L)				Mean measured concentrations (mg/L)
	t=0h	t=24h	t=48h	t=72h
0 (control)	n.d.	n.d.	n.d.	n.d.	n.a.
1.0	0.710	0.464	0.0918*	0.224	0.41*
3.2	3.95	2.585	1.30	1.19	2.0
10	7.86	5.103	3.55	2.43	4.3
10 (without algae)	8.41	5.449	3.78	2.15	4.4
32	26.6	15.2	8.60	6.258	12
100	80.6	10.8*	25.8	16.3	37*

*  These values were measured not in exposure vessels but in vessels set for analysis of test concentrations. Therefore, these measurements were excluded from calculations and for these two WAFs time weighted average concentrations were calculated.

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'.

Conclusions:

The ErC50, ErC10 and NOEC were 26, 12 and 4.3 mg/L, respectively.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Water Accommodated Fractions (WAFs) were prepared in closed vessels at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L applying a 2-day period of magnetic stirring followed by a one-hour stabilisation period. The aqueous fractions were collected by siphoning and used as test solutions. The final test solutions were all clear and colourless. Pseudokirchneriella subcapitata was exposed for 72 hours (three replicate flasks per concentration) under constant illumination and shaking at a temperature of 22 - 23°C. Samples were taken from all treatments at t = 0 , 24, 48 and 72 h and analysed with a validated GC-FID method. At the start of the test, the measured test concentrations were generally in agreement with the loading rates. The measured concentrations decreased to 20 - 32 % of the initial concentrations at the end of the test and therefore the measured concentrations were used for calculation of effect concentrations. Loading rates of 1.0, 3.2, 10, 32 and 100 mg/L corresponded with mean measured concentrations of 0.41, 2.0, 4.3, 12 and 37 mg/L, respectively. Statistically significant inhibition of growth rate was found at mean measured concentrations of 12 mg/L and higher. The ErC50, ErC10 and NOEC were 26, 12 and 4.3 mg/L, respectively.

Description of key information

Data on the toxicity to aquatic algae is available from a study according to OECD 201 and the 72-h ErC50 and 72-h ErC10 values determined at 26 mg/L and 12 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

26 mg/L

EC10 or NOEC for freshwater algae:

12 mg/L

Additional information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria (IFF, 2015). In this study, exponentially growing freshwater green alga (P. subcapitatus) were exposed to nominal test substance concentrations of 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours under static conditions. The test was performed in 3 replicates per test concentration. In 2 separate replicates the nominal test concentrations were analytically verified at t=0h, t=24h, t=48h and t=72h. Geometric mean measured concentrations were determined to be 0.41, 2.0, 4.3, 12 and 37 mg/L. At the start of the test and after 24, 48 and 72 hours exposure duration, the algae were scored for number of cells and for microscopic abnormalities. A statistically significant inhibition of growth rate of ca. 10% was observed at a concentration of 12 mg/L. This effect increased to ca. 73% at the highest tested concentration of 37 mg/L. Based on these findings, the 72-h ErC50 and 72-h ErC10 values are determined at 26 mg/L and 12 mg/L, respectively. The 72 -h NOEC is 4.3 mg/L. All effect values are expressed as measured concentrations.