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DL-LACTONE CAS N°:79-50-5 - page 44 / 113





44 / 113



ID: 79-50-5 DATE: 18.01.2006

Test substance:

as prescribed by 1.1 - 1.4


Test substance concentrations dl-Lactone was tested in duplicate at 43 mg per 2 litres, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. Furthermore, an additional adsorption control bottle was prepared at 100 mg dl-Lactone/l. Since dl-Lactone was easily soluble in water the test media were prepared using a stock solution of 1 g/l in milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). A weighed amount of 1006.7 mg of dl-Lactone was dissolved in milli-RO water and made up to 1000 ml. The stock was a clear and colourless solution. Aliquots of 43 ml of the stock solution were added to the test medium, containing the microbial organisms, of test substance bottles A and B, toxicity control, the abiotic control and the adsorption control. An aliquot of 200 ml of the stock solution was added to the test medium of the additional adsorption control, resulting in a final dl-Lactone concentration of 100 mg/l. All test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

Reference substance: A solution of sodium acetate (>=99%, Merck) was prepared by dissolving 202.4 mg in Milli-RO water and making this up to a total volume of 50 ml. Volumes of 20 ml from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg/l sodium acetate (12 mg TOC/l). Test system Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands, receiving predominantly domestic sewage. Treatment: The sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.9 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). The sludge was coarsely sieved and washed twice with tap-water. After washing the sludge was made up to the original volume. A small amount of the sludge was weighed and dried at approximately 105 °C to determine the amount of suspended solids. An amount of the sludge corresponding to approximately 30 mg/l suspended solids was added to the mineral medium, except for one bottle, an additional adsorption control, in which the amount of the sludge corresponded to approximately 1000 mg/l suspended solids. On the day the sludge was sampled, a rough indication on the concentration of suspended solids was made (drying for 4 hours). This indication was used for the addition of the sludge to the test bottles. The exact amount of suspended solids was determined after drying overnight. Colony count: From the supernatant of the sludge the heterotrophic microbial colony count was determinedon agar plates (diameter 9 cm), which contained purified agar (Oxoid Ltd, UK, 18 g/l) and nutrient broth (Oxoid Ltd, UK, 25 g/l). Test procedure and conditions Test duration: 28 days (last CO2-measurement on the 29th day).



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