ISO 13641-2:2003 pdf download

ISO 13641-2:2003 pdf download.Water quality -Determination of
inhibition of gas production of anaerobic bacteria — Part 2: Test for low biomass concentrations.
However, if the seals of the bottles are reliable for only one or a few piercings, set up a batch preferably in triplicates of test bottles for each time interval (t) for which results are required for all mass concentrations of a test material to be tested. Similarly set up “t” batches of bottles for the reference substance and for the controls.
The use of a glove box (5.9) is recommended. At least 30 mm before starting the test, let nitrogen flow into the glove box containing all necessary test equipment. If a glove box is not used, de-gas the bottles using nitrogen for air displacement. Make sure that the temperature of the inoculum corresponds to the incubation temperature (6.1) during the operation and while the bottles are sealed.
7.1.2 Preliminary test
If the activity of the inoculum (4.2.1) is unknown, it is recommended to carry out a preliminary test. Set up controls to give, for example, mass concentrations of solids of 0,1 g/l, 0,2 g/l and 0,4 g/l plus substrate but use no test material. Also, use different volumes of reaction mixture in order to have 3 or 4 different ratios of volume of headspace to volume of liquid. Measure biogas in regular intervals. From the results of biogas produced the most suitable conditions for the main test can be deduced which allow the yield of sufficient biogas for measurements and hence the optimal sensitivity without the fear of explosions. Using results from the preliminary test, select the frequency at which pressure measurements should be made, the test duration and the need of acidification at the end of the test.
7.2 Test materials and controls
7.2.1 Test materials
7.2.1.1 Test compound solutions
Prepare separate stock solutions for each water-soluble test compound in oxygen-free dilution water (4.1.1) to contain, for example, 10 g/l of test material. Use appropriate volumes of these stock solutions to prepare the reaction mixtures containing graded mass concentrations. Alternatively prepare a dilution series of each stock solution so that the volume added to the test bottles is the same for each required final mass concentration.
Add substances with little or no water-solubility, for example, as solutions in a volatile solvent. Prepare such a solution at an appropriate mass concentration in a suitable solvent, for example, acetone or diethyl ether (but do not use inhibitory solvents such as trichloromethane or tetrachloromethane). Add the solutions to the empty test bottles (5.6) and evaporate the solvent before the addition of the inoculum. Liquid water-insoluble test materials may be injected directly into inoculated serum bottles using microsyringes (5.7). For other treatments, see for example ISO 10634, but be aware that any surfactants used to produce emulsions can be inhibitory to anaerobic biogas production.
Add test compounds to the bottles (5.6) to give a geometric series of mass concentrations, for example,
500 mg/I, 250 mg/I, 125 mg/I, 62,5 mg/I, 31,2 mg/I and 15,6 mg/I. If the range of the toxicity is not known from
similar compounds, carry out a preliminary range-finding test with mass concentrations of, for example,
1 000 mg/I, 100 mg/I and 10 mg/I so as to ascertain the appropriate range.
7.2.1.2 Waters and wastewaters
Use the original sample of waters and wastewaters as stock solution, and, if necessary, adjust the pH to 7 ± 0,5 if inhibition due to an acidic or alkaline sample is not to be determined.
Add waters and wastewaters to the bottles (5.6) to give a geometric series of final dilution steps as follows: 1:2, 1:4, 1:8, 1:16 and so forth, where these dilution ratios are expressed as volume of water or wastewater to the total end-volume.
When testing wastewater, the highest possible test mass concentration corresponds to 50 % of the wastewater sample. It results by adding the original wastewater to the test bottles (5.6) and an equal volume of the inoculum suspension. Make sure that wastewaters or other test waters are sufficiently free of oxygen. For example, bubble nitrogen gas (4.1.2) through the dilutions for at least 1 h and use oxygen-free dilution water (4.1.1).