Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate

Toxicity to microorganisms

Description of key information

The toxicity of the test substance to microorganisms was determined in an activated sludge respiration inhibition test according to OECD TG 209 in compliance with GLP. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007). The batch of test substance tested was a clear colourless liquid with a purity of 94.1%. No correction was made for the purity/composition of the test substance. Preparation was as much as possible performed under yellow light and/or low light conditions. The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 mL of test substance mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. In a first experiment the mixtures were stirred in closed dark brown bottles for 24-26 hours. Subsequently, 16 mL synthetic medium, 250 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates. Since the test substance had some volatile properties, it was decided to perform two additional experiments. The preparation method in the 2nd and 3rd experiment was comparable to the 1st experiment, except that the loading rates were not stirred before addition of synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour (experiment 1 and 2) or 30 minutes (experiment 3) exposure period. Thereafter, oxygen consumption was recorded for 10-11 minutes. Experiment 1 (test substance loading rates stirred for 24-26 hours before the 3-hour exposure period) showed 9, 25 and an average of 31% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg CP Formate/l, respectively. Experiment 2 (test substance loading rates not stirred before the 3-hour exposure period) showed no inhibition of the respiration rate at 10 and 100 mg test substance/L and an average of 34% inhibition of the respiration rate at a loading rate of 1000 mg/L. Experiment 3 (test substance loading rates not stirred before the 30-minutes exposure period) showed no inhibition of the respiration rate at 10 mg test substance/L and 10% and an average of 36% inhibition of the respiration rate at a loading rate of 100 and 1000 mg/L, respectively. In all experiments, the inhibition observed at a loading rate of 1000 mg/L was statistically significant (Two Sample t-Test). Since, all test results were comparable it appears that the compound has not evaporated from the test media during the test substance preparations and/or exposure. There was no oxygen uptake from abiotic processes and the results at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The study met the acceptability criteria prescribed by the protocol and was considered valid. In conclusion, test substance was slightly toxic to waste water (activated sludge) bacteria. The EC50 exceeded a loading rate of 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L