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DL-LACTONE CAS N°:79-50-5 - page 63 / 113





63 / 113




ID: 79-50-5 DATE: 18.01.2006

continuous shaker in a climatised (22±0.5 °C) room. The pH was measured at the beginning and end of the test. Range-finding test: A range-finder was performed with 4 concentrations of 100, 10, 1 and 0.1 mg/l. Main test: Based on no observed effects in all concentrations in the pretest, a limit test with 6 replicates at 100 mg dl-lactone/l and 6 replicates of blank (medium only) control was performed. Additionally, 2 replicates of 100 mg dl-lactone/l without algae and 1 extra replicate of both concentrations (100 and 0 mg/l) necessary for sampling were made up. Cell densities: At the beginning of the test, cell density was counted using a microscope counting chamber. In parallel and thereafter, cell densities were determined by spectrophotometry at 720 nm (details in report). Sampling and analytics: Samples of 10 ml each were taken from the blank controls and the 100 mg/l solutions at 0, 24 and 72 hours after first introduction into test media. To follow the actual test substance concentration over time, a test vessel at 100 mg/l but without algae was also sampled at the start and end of the test period. Samples were analysed by HPLC immediately, without storage. HPLC conditions


LiChrospher 100RP-18, 250*4 (i.d.) mm, d(rho)= 5 µm (Merck, Germany)

Mobile phase

20/80/0.1 (v/v/v) acetonitrile/Milli-Q

Flow Detection


water/formic acid 1 ml/min SCIEX MSMS system API-300 mass spectrometer (Perkin Elmer, USA) ion-spray, positive mode

Monit masses

Inject volume Int standard

MRM m/z 131.3 --> 113.0 (test substance) MRM m/z 127.2 --> 98.9 (internal standard) 100 µl 4-hydroxy-6-methyl-2-pyrone (98%, Sigma- Aldrich, USA)

Data handling


Quantification of cell densities was based on a calibration curve of counted cell density versus extinction from six different cell densities. this correlation served to determine cell densities at the various time points in the test. NOEC, areas under the growth curve, comparison of growth rates and calculation of EbC50 and ErC50 values were made as recommended in the OECD guideline 201. In the range-finding test, no significant effects were noted up to a concentration of 100 mg/l (full data in report). Therefore, a limit test scheme was adopted for the main test. In the limit test, the concentration remained above 80% of the measured initial concentration of 105 mg/l during the first 24 h. At 72 h, the concentration in the sample with the algae had decreased from 105 mg/l to 45 mg/l while in the nominal 100-mg/l solution without algae it had remained at 76 mg/l. The presence of an extra peak in the chromatograms indicated that the decrease in the algal solution was probably related to degradation, which had not happened in the algae-free solution. Based on the measured concentrations in the algal suspensions, the average measured exposure concentration was



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