2,4,6-trimethylcyclohex-3-enecarbaldehyde

Toxicity to aquatic algae and cyanobacteria

Description of key information

The toxicity of the test substance to aquatic algae was assessed according to OECD Test Guideline 201.  The 72 hour EC50 based on growth rate was 3.0 mg/L with 95 % confidence limits of 2.8 – 3.2 mg/L.  The EC10 was 1.1 mg/L, the NOEC was 0.45 mg/L and the LOEC was 1.1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

3 mg/L

EC10 or NOEC for freshwater algae:

1.1 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

 

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication.

 

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 85 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

 

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 2.6, 6.4, 16, 40 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately
1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.30 to 50 mg/L. Measured concentrations at 72 hours were in the range of 0.68 to 46 mg/L. Analysis of samples at 72 hours prepared with the omission of algal cells and incubated alongside the test showed measured test concentrations to range from 0.50 to 44 mg/L. Given this it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

The 72 hour EC50 based on growth rate was 3.0 mg/L with 95 % confidence limits of 2.8 – 3.2 mg/L. The corresponding EC10 was 1.1 mg/L, the NOEC was 1.1 mg/L and the LOEC was 3.0 mg/L.

The preferred observational end point in this study is algal growth rate inhibition 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.