3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

EC Number:

276-650-6

EC Name:

3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

Cas Number:

72403-67-9

Molecular formula:

C15H24O2

IUPAC Name:

[3-(4-methylpent-3-en-1-yl)cyclohex-3-en-1-yl]methyl acetate; [4-(4-methylpent-3-en-1-yl)cyclohex-3-en-1-yl]methyl acetate

Test material form:

other: liquid

Details on test material:

Test substance name: Myraldyl acetate
CAS number: 72403-67-9
Molecular formula: C15H24O2
Molecular weight: 236.3
Vapour pressure: 0.046 Pa at 20 ºC
Water solubility: 2.50 mg/L at 20 ºC
Octanol water partition co-efficient (Log Pow): 5.6 to 5.7
Description: Colourless liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analysis of the test media samples was conducted at 0, 24, 48 (with algae) and 72 hours (with and without algae).

Test solutions

Details on test solutions:

The test substance was known to have limited solubility in water (2.50 mg/L at 20 ºC)

Initial visual solubility work was conducted which indicated that the highest attainable concentration was 1.0 mg/L using an initial solvent spike method of preparation.

A media preparation trial indicated that the most appropriate method of preparation for the test substance was the use of a saturated solution method of preparation. Analysis of a saturated solution prepared from an initial 100 mg/L loading rate followed by filtration gave a measured concentration of 0.97 to 1.21 mg/L indicating this to be the limit of solubility of the test item in algal media.

The test substance was therefore prepared from an initial 100 mg/L, stirred for ca 24 hours and filtered to give a nominal test concentration of 100% saturated solution.

Preparation of Test Medium
The dilution media used was EC medium. Full preparation details are presented in Appendix 3.
At the start of the test, the 100% saturated solution test concentration was prepared by weighing ca 200 mg of test substance and adding to 2000 mL of EC medium. This was stirred for ca 24 hours and filtered through a 0.45 μm filter to give the 100% saturated solution test concentration. Dilutions were then prepared in EC medium to give the remainder of the nominal test concentrations of 10, 18, 32 and 56% saturated solution. A control treatment was prepared by adding EC medium only to the control vessels.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test organism, Pseudokirchneriella subcapitata (Strain 278/4), was originally obtained from the Culture Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton. This species is recommended for testing in accordance with the OECD regulatory guidelines.
Prior to testing, duplicate starter cultures were prepared and incubated under test conditions (as detailed in Appendix 3) to obtain sufficient algal cells in exponential growth and to achieve a starting alga cell density of 1 x 104 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Test temperature:

The laboratory temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital temperature logger.

pH:

At the start of the test, the pH of freshly prepared test media, before and after alga cell inoculation was determined. The pH in each test vessel was also determined at the end of the test.

Salinity:

No (freshwater)

Nominal and measured concentrations:

Range finding test:
Nominal concentrations: 0.10, 1.0, 10 and 100% saturated solution

Definitive test:
Nominal concentrations: 10, 18, 32, 56 and 100% saturated solution,
Geometric mean measured concentrations: 0.13, 0.23, 0.41, 0.90 and 1.2 mg/L

Details on test conditions:

Range-finding Test
The range-finding test was conducted at nominal test substance concentrations of 0.10, 1.0, 10 and 100% saturated solution. A control group was also included. Duplicate test vessels were prepared for the control and each test concentration. Based on nominal concentrations, the results of the range-finding test suggested that the 72-hour ExC50 value would be between 10 and 100% saturated solution.

Definitive Test
Based on the results of the range-finding test, for which only the key findings have been reported, the definitive test was conducted at nominal test concentrations of 10, 18, 32, 56 and 100% saturated solution (0.13, 0.23, 0.41, 0.90 and 1.2 mg/L based on geometric mean measured concentrations of the 0 and 24 hour analytical results).

Environmental Conditions
All flasks were loosely-capped and incubated under a light bank in a temperature controlled laboratory. The vessels were placed on an orbital shaker (ca 100 rpm) under conditions of constant light (4440 to 8880 Lux), using cool white fluorescent tube lights, emitting white light across the visible portion of the spectrum (400 - 700 nm).

At the start of the test, the pH of freshly prepared test media, before and after alga cell inoculation was determined. The pH in each test vessel was also determined at the end of the test.

The laboratory temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital temperature logger.

The light intensity within the incubator was monitored at the start and end of the test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.91 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.66-0.94

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Effects based on yield and biomass were also reported (see attached full study report and summary below). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table.

Based on geometric mean measured test concentrations from the 0 and 24 hour analytical results, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 0.98, 0.96 and >1.2 mg/L, respectively.
The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 0.41 mg/L.

Validity Criteria
The control cell densities increased by a factor of 93. Therefore the validity criterion of achieving an increase in control cell density by at least a factor of 16 was achieved, and the environmental conditions of the test were considered acceptable. The mean coefficient of variation for section-by-section specific growth rates (0-24, 24-48 and 48-72 hours) in the control group was 3.44% and therefore did not exceed 35%.

The coefficient of variation for average specific growth rates during the whole test period in replicate control group was 2.31% which did not exceed 7% and therefore the validity criteria were met.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effects of myraldyl acetate on the growth of the unicellular green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011). All validity criteria were met therefore the test was considered valid.
The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be > 1.2 mg/L and 0.91 mg/L respectively.