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1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested for its acute toxicity in fish, the study procedures described were based on the ISO International Standard 7346-1: Static method, 1996. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.l ,1992, the OECD guideline No. 203, 1992 and the OECD series on testing and assessment number 23, 2000. The batch of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE tested was a light yellow viscous liquid with a purity of 99.0% and completely soluble in test medium at the concentrations tested. Preparation of the test solution started with a stock solution of 100 mg/L applying 16 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The final test solution was clear and colourless. A limit test was performed, exposing seven fish per concentration to a blank-control and a nominal concentration of 100 mg/L. The total test period was 96 hours and samples for analytical confirmation of actual test concentrations were taken from the blank-control and the limit concentration at the start, after 24 hours of exposure and at the end of the test period. Analysis of the samples taken at the start of the limit test showed that the mean actual test concentration was in agreement with nominal (104%) and remained stable during the 96-hour test period (i.e. 102% of initial). Based on these results, the NOEC and the LC50 were based on nominal concentrations. The study met the acceptability criteria prescribed by the protocol and was considered valid. 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE induced no visible effects in carp at or below 100 mg/L (NOEC). The 96h-LC50 was > 100 mg/L based on analytically confirmed nominal concentrations.

This is also supported by an acute fish toxicity study in zebra fish, showing that 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE induced no mortality up to the highest concentration of 150 mg/L.

Furthermore an acute Toxicity study in daphnids was performed, the study procedures described were based on the ISO International Standard 6341, 1996. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.2, 1992 and the OECD guideline No. 202 Part I, 1984.

The batch of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE tested was a light yellow viscous liquid with a purity of 99.0% and completely soluble in test medium at the concentrations tested. Preparation of test solutions started with a stock solution of 100 mg/L applying 15-20 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless. The study started with a range-finding test, exposing ten daphnids per concentration (in duplicate, 5 per vessel) to a range of 0.1 to 100 mg/L increasing by a factor of 10 and to a blank-control. No immobility was observed in any of the test solutions during the test period. The analytical results showed a mean measured concentration of 102 mg/L at a nominal concentration of 100 mg/L at the start of the test. Further, this mean measured concentration proved to be stable under test conditions for at least 48 hours. Based on these results, the expected 48h-EC50 was above a concentration of 100 mg/L. The study was continued with a limit test, exposing twenty daphnids per concentration (in quadruplicate, 5 per vessel) to a blank-control and a nominal concentration of 100 mg/L. The total test period was 48 hours and samples for analytical confirmation of actual test concentrations were taken from the blank-control and the limit concentration at the start and the end of the test period. Analysis of the samples taken at the start of the limit test showed that the mean actual test concentration was in agreement with nominal (99%) and remained stable during the 48-hour test period (i.e. 98% of initial). Based on these results, the NOEC and the ECso were based on nominal concentrations. The study met the acceptability criteria prescribed by the protocol and was considered valid. 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE did not induce acute immobilisation of Daphnia magna at 100 mg/L after 48 hours of exposure (NOEC). The 48h-EC50 was > 100 mg/L based on analytically confirmed nominal concentrations.

1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was also tested for potential toxicity towards aquatic algae, the study procedures described were based on the ISO International Standard 8692, 1989. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.3, 1992, the OECD guideline No. 201, 1984 and the OECD series on testing and assessment number 23, 2000.

The batch of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE tested was a light yellow viscous liquid with a purity of 99.0% and completely soluble in test medium at the concentrations tested. Preparation of test solutions started with a stock solution of 100 mg/L applying 17 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless.

A combined limit/range-finding test was performed, exposing six replicates of exponentially growing algal cultures to a nominal 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE concentration of 100 mg/L and a blank-control. In addition three replicates of exponentially growing algal cultures were exposed to nominal 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE concentrations of 0.1, 1.0 and 10 mg/L. The total test period was 72 hours and samples for analytical confirmation of actual test concentrations were taken from the blankcontrol and the 100 mg/L concentration at the start and the end of the test period. Analysis of the samples taken at the start of the combined limit/range-finding test showed that the mean actual test concentration was in agreement with nominal (86%) and remained stable during the 72-hour test period (i.e. 102% of initial). Based on these results, the NOEC and the EC50 were based on nominal concentrations. The study met the acceptability criteria prescribed by the protocol and was considered valid.

1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE inhibited cell growth of Selenastrum capricornutum significantly (29%) at nominally 100 mg/L. However, no reduction of growth rate was recorded at any of the concentrations of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE tested. The EC50 for cell growth inhibition (EbC50: 0-72h) was beyond 100 mg/L based on analytically confirmed nominal concentrations.

As growth rate is derived from the slope under the growth curve in a logarithmic plot, the´measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth.

The EC50 for growth rate reduction (ErC50: 0-72h) was beyond 100 mg/L based on analytically confirmed nominal concentrations.

The NOEC for cell growth inhibition was <100 mg/L. The NOEC for growth rate reduction was 100 mg/L.

The influence of1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE on the respiration rate of activated sludge was investigated after a contact time of 30 minutes. The study procedure was based on OECD Guideline No. 209, adopted April 4, 1984, EEC Directive 67/548 amended November 18, 1987 (87/302), Part C, Publication No. L133, adopted May 30, 1988 and ISO Standard 8192 (1986). 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was a light yellow viscous liquid with a purity of 99.0% (HPLC). The water solubility of1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE is > 1000 g/L at 20.1 ± 0.5 °C (pH 8.3, NOTOX Project 4"17206). For the activated sludge respiration inhibition test a stock solution of 0.5 g/L was prepared by adding 508.3 mg test substance to 1000 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Thorough mixing (10 minutes) was used to accelerate dissolving and to ensure homogeneity. The pH of the clear and colourless stock solution was 7.7. A concentration of 100 mg/L, corresponding to 100 ml of the prepared stock solution in 500 ml final volume, was tested in duplicate. 11 % inhibition of respiration rate of the sludge was recorded at 100 mg 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Since all criteria for acceptability of the test were met, this study was considered to be valid. In conclusion, under the conditions of this present test, 11 % inhibition of respiration rate of the sludge was recorded at 100 mg 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE per litre. Hence, the EC50 of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE exceeded 100 mg/L.