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DL-LACTONE CAS N°:79-50-5 - page 45 / 113





45 / 113



ID: 79-50-5 DATE: 18.01.2006

During the test period the test media were aerated and stirred continuously. Test vessels: 2 litre all-glass brown coloured bottles. Milli-RO/Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion exchange cartridges (Milli-Q; Millipore Corp., Bedford, Mass., USA). Stock solutions for reconsituting water from Milli-Q or Milli-RO water were as per the OECD guideline. Such water was stored in a sealed vessel to prevent absorption of CO2 from the air. Synthetic air (CO2 < 1 ppm): A mixture of oxygen (21%) and nitrogen (79%) was passed through a bottle, containing 0.5-1 l 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min). Preparation of bottles Pre-incubation medium: Mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2. Type and number of Test suspension: containing test substance bottles and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles). Procedure control: containing reference substance and inoculum (1 bottle). Toxicity control: containing test substance, reference substance and inoculum (1 bottle). Abiotic control: containing test substance and sterilising agent (1 ml/l of a solution containing 10 g/l of HgCl2; 1 bottle). Adsorption control: containing test substance, inoculum and sterilising agent (1 ml/l of a solution containing 10 g/l of HgCl2; 1 bottle). Additional adsorption bottle: Additional adsorption control (only used for DOC analyses): containing test substance at 100 mg/l and inoculum at 1000 mg/l (1 bottle). This bottle was not in series with the other bottles and not connected to CO2-absorbers. Preparation: The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume). The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. Determination of CO2 Test bottles: All test bottles, except the additional adsorption bottle. Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of

CO2 produced was determined by titrating the

remaining Ba(OH)2 with 0.05 M from 1 M HCl (Titrisol ampul; Measurements: Titrations were

standardized HCl (1:20 dilution Merck KGaA, Germany). made every second or third day

during the first 10 days and day until the 28th day. Each

thereafter at least every fifth time the CO2-absorber nearest to



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