1、May 2009DEUTSCHE NORM English price group 14No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 13.060.70!$W_ 3 mg/l is re
2、quired for the test. If the oxygen concentration of the undiluted sample is less than 3 mg/l, use adequate methods to oxygenate the sample, e.g. aeration or stirring. Measure the pH of all samples. If the pH is between 6,0 and 8,5, an adjustment is usually not necessary. Adjustment of the pH-value,
3、however, may alter the nature of the sample. On the other hand, the pH of the sample and the pH of the test batch may differ because of the buffer capacity of the test medium. It may be necessary to carry out tests on both the pH-adjusted and the non-pH-adjusted samples. If necessary, adjust the pH
4、of the sample by adding either hydrochloric acid (5.4) or sodium hydroxide solution (5.3). Depending on the purpose of the test, the pH may be adjusted to 7,0 0,2 or to the upper (8,5 0,2) and lower limits (6,0 0,2). Choose the concentration of the hydrochloric acid or the sodium hydroxide solution
5、to restrict the volume added to not more than 5 % of total volume. Add 20 g of sodium chloride per litre to the water sample or to the neutralized water sample. For samples with high salt concentrations, measure the salinity and add the amount of salt which is necessary to adjust the osmolarity to 2
6、0 g/l NaCl. If the sample contains between 20 g/l and 50 g/l NaCl-equivalents, add no salt. The resulting salt-concentration in the test samples shall not exceed the osmolarity of a 35 g/l sodium chloride solution. For salt water samples, Annex D gives further information. Strongly turbid samples sh
7、ould be allowed to settle for 1 h or centrifuged, for example for 10 min at 5 000g, or should be filtered. Use the supernatant or filtrate for the test. 8 Procedure Prepare the reference samples according to 5.6. Test each batch of bacteria after delivery with all three reference substances. Test at
8、 least one of the three reference substances in parallel with each stock-suspension test tube thawed for the tests. Prepare the samples according to 7.2. Thaw the liquid-dried bacteria (stock suspension) in a water bath at 20 C 2 C. Refrozen stock suspensions may be used for preliminary tests only.
9、DIN EN ISO 11348-2:2009-05 EN ISO 11348-2:2008 (E) 8Prepare the test suspension from the stock suspension in two steps: Add 0,5 ml (per 100 l stock suspension in the test tube) of solution (5.5), maintained at 15 C 1 C, and homogenize by gentle shaking of the test tube. Wait for about 15 min. Pipett
10、e this suspension into a reagent vessel (approximately 20 ml volume) and add 11,5 ml of solution (5.5), maintained at 15 C 1 C, and homogenize by gentle shaking of the reagent vessel. Wait for about 15 min. Prepare, in a first set of test tubes (6.4), the sample dilution series, the reference sample
11、 (5.6) and the controls (5.2) required. A common procedure for the preparation of the dilution series is described in Annex B. Depending on the purpose of the test and the statistical requirements concerning the test results, other dilution designs with concentrations in a geometric or a logarithmic
12、 series may be appropriate as well. Due to mixing of equal volumes of sample/diluted sample and test suspension, the highest sample concentration in the test is 50 % sample as a rule. For the testing of nearly undiluted water samples (80 % sample), an extra control batch is needed (see B.2 and Table
13、 1). Maintain the test tubes containing the sodium chloride solution (5.2) for controls, the reference samples (5.6), the samples (7.2) and the samples of the dilution series (Table B.1) at 15 C 1 C. Chose test conditions which safeguard that the maximum temperature deviation in the thermo-block wit
14、hin one test is at most 0,3 C. For tests with equal volumes of test suspension and sample, pipette 500 l portions of the test suspension into a second, corresponding set of test tubes (6.4), maintained at 15 C 1 C in the incubator, at the same time intervals (5 s to 20 s) as used for later intensity
15、 measurements. Carry out two parallel determinations per dilution level at a test temperature of 15 C 1 C. Adjust the luminometer instrument to a convenient, near-maximum setting. Determine and record the luminescence intensity, I0, of the test suspensions by means of a luminometer. As the contact t
16、ime for all test samples shall be equal, use a chronometer (6.6) for the measurement of the luminescence intensities at equal time intervals, seriatim. An interval of 5 s to 20 s has been found convenient. Measure all test suspensions, as differing luminescence may be expected due to possible inhomo
17、geneities of the test suspension. Immediately after the initial luminescence measurement of a test suspension, make up this suspension to a total volume of 1 ml with samples (7.2), diluted samples (Annex B), reference sample (5.6) or sodium chloride solution (5.2), as appropriate. This is done by pi
18、petting 500 l each of samples (7.2), diluted samples (Annex B), reference sample (5.6) or sodium chloride solution (5.2), prepared in the first set of test tubes, to the test suspensions in each of the tubes in the corresponding second set of test tubes. Mix by hand, start the chronometer and place
19、the test tubes back into the thermo-block at 15 C 1 C. Repeat for all the other test tubes, leaving the same time interval between successive additions. Determine and record the luminescence intensity in all test tubes of the second set of test tubes, including controls, after, optionally, 5 min (I5
20、) and again after 15 min and 30 min (I15, I30), as required, at intervals of 5 s to 20 s. Record the instrument adjustment. DIN EN ISO 11348-2:2009-05 EN ISO 11348-2:2008 (E) 99 Evaluation 9.1 Inhibitory effect on luminescent bacteria Calculate the correction factor (fkt-value) from the measured lum
21、inescence intensity using Equation (1). This factor serves to correct the initial values I0of all test samples before they can be used as reference values for the determination of the water-dependent decrease in luminescence. fkt= Ikt/ I0(t = 5 min, 15 min, 30 min) (1) where fktis the correction fac
22、tor for the contact time of 5 min, 15 min or 30 min; Iktis the luminescence intensity in the control sample after the contact time of 5 min, 15 min or 30 min, in relative luminescence units; I0is the luminescence intensity of the control test suspension, immediately before the addition of the diluen
23、t (5.2), in relative luminescence units. Calculate the mean correction factor ktf and the deviation of the individuals from the means in percent (one significant digit): ()kkik/ 100tt tfff(2) where fktiis either of the two individual values of the correction factor and ktf is the mean value. Calcula
24、te Ictusing Equation (3): ct 0 ktI If= (3) where ktf is the mean of fkt; I0is the luminescence intensity of the test sample suspension, immediately before the addition of the sample (7.2) or the diluted sample (Annex B), in relative luminescence units; Ictis the corrected value of I0for test sample
25、tubes immediately before the addition of the test sample. Calculate the inhibitory effect of a test sample using Equation (4): ()/ct ct100ttHIII= (4) where Htis the inhibitory effect of a test sample after the contact time of 5 min, 15 min or 30 min, in percent; Ictsee Equation (3); Itis the lumines
26、cence intensity of the test sample after the contact time of 5 min, 15 min or 30 min, in relative luminescence units. Calculate the mean of the inhibitory effect Htfor each dilution level, in percent. DIN EN ISO 11348-2:2009-05 EN ISO 11348-2:2008 (E) 10Calculate the arithmetic difference of the par
27、allel determinations of Htifrom their respective mean ,tH in percent points (one significant digit): i(%) (%)ttHH where Htiis either of the two individual values of the inhibitory effects of a test sample and tH is the mean value. 9.2 Determination of EC-values Calculate the concentration-effect rel
28、ationship for each exposure time using suitable standard linear or non-linear regression analysis8. For evaluation of concentration-effect relationships using a linear regression technique, evaluate, for each dilution level, the gamma value (ratio of light lost to the amount of light remaining at ti
29、me t) using Equation (5): ()/ 100tttHH=(5) where tis the gamma value of the test sample after the contact time of 5 min, 15 min or 30 min; tH is the mean of Ht, see Equation (4). NOTE When a certain test concentration gives 0 % or 100 % inhibition of bioluminescence, the gamma value cannot be calcul
30、ated. Therefore usually only Ht-values between 10 % and 90 % are used in the calculation of the concentration-effect relationship. The concentration-effect relationship at a given exposure time often can be described by the following linear Equation (6): lg ct= b lg t+ lg a (6) where ctis the portio
31、n of the water sample within the test sample, in percent; tsee Equation (5); b is the value of the slope of the described line; lg a is the value of the intercept of the described line. By means of standard least-squares regression statistics, calculate the EC20- and EC50-values with corresponding c
32、onfidence limits, in which: ct= EC20,tat t= 0,25; ct= EC50,tat t= 1,00. For non-linear regression analysis various models are available within standard graphic or statistical software packages. They are typically based on functions of the normal distribution (i.e. Probit analysis), the logistic dist
33、ribution (i.e. Logit analysis), or the Weibull distribution (i.e. Weibull analysis). Calculated inhibitory effects (Ht) can be used directly to estimate parameters of the nonlinear concentration-effect relationship, from which EC-values for any level might subsequently be derived8. DIN EN ISO 11348-
34、2:2009-05 EN ISO 11348-2:2008 (E) 11If the range of value pairs cannot be curve-fitted, the EC-values can be estimated graphically using a double logarithmic coordinate system. 10 Expression of results Report the results in accordance with the example in Table 1. Report the test duration (5 min, 15
35、min or 30 min). If determined, report the LIDlb-value (see Annex B). If determined, report the EC20- and EC50-values and the method for the derivation of these values. Report the type of bacterial preparation used. DIN EN ISO 11348-2:2009-05 EN ISO 11348-2:2008 (E) 12Table 1 Example of test evaluati
36、on Sample: effluent from a sewage treatment plant Control experiments Control batch number Dilution level D Measured values k30 0/I I k30f 0I k30I Validity test Deviation from the mean k30f in %b1 1a93 76 0,817 2 0,815 2 0,3 2 91 74 0,813 2 3 W 2a92 79 0,858 7 0,850 4 1,0 4 95 80 0,842 1 Test experi
37、ments Test batch number Dilution level D Measured values c30I 30H 30H Validity test Deviation from the mean, in % pointsc30 0I 30I % % 1 1 92 25 75,0 66,7 65,53 1,1 1,901 2 93 27 75,8 64,4 3 2 86 43 73,1 41,2 42,51 1,3 0,740 4 90 43 76,5 43,8 5 3 91 60 77,4 22,5 22,92 0,5 0,297 6 89 58 75,7 23,4 7 4
38、 95 72 80,8 12,4 11,65 0,8 0,132 8 94 70 79,9 10,9 Reference substance 9 4,5 mg/l DCP, or 25 mg/l Zn, or 4 mg/l Cr 91 32 77,4 58,7 57,85 0,9 1,372 10 93 34 79,1 57,0 aSee Annex B. bFor the control batch, the deviation from the mean k30f is determined by the arithmetic difference of the parallel dete
39、rminations from the mean, divided by the mean expressed in percent Equation (2) in 9.1. cFor the test batch, the deviation of the H30-values (in percent) of the parallel measurements from the mean is calculated as the arithmetic difference of each H30-value (in percent) from the mean 30H (in percent
40、) (called percent points). The LIDlb-value in this example is LIDlb= 4. The EC20-value in this example is 31,9%; the EC50-value is 58,8 % (standard least square statistics). DIN EN ISO 11348-2:2009-05 EN ISO 11348-2:2008 (E) 1311 Criteria of validity The test is valid if: the ktf value for 15 min or
41、 30 min incubation ranges between 0,6 and 1,3; the parallel determinations do not deviate from their mean by more than 3 % for the control samples; for the test samples which determine the LIDlb-value or the EC20/EC50-values, respectively, the deviation from their mean in “percent points” does not e
42、xceed 3 % points (see Note c in Table 1); for the batch of bacteria delivered, the three reference substances (5.6) (solutions not neutralized, check separately) cause 20 % to 80 % inhibition after 30 min contact time at the following concentrations in the final test suspension: 4,5 mg/l 3,5-dichlor
43、ophenol 25 mg/l Zn(II) (equivalent to 109,9 mg/l zinc sulfate heptahydrate) 4 mg/l Cr(VI) (equivalent to 11,3 mg/l potassium dichromate); one of these three reference substances (5.6) (solution not neutralized) tested in parallel to each stock suspension test tube thawed for the actual test (see Cla
44、use 8) causes 20 % to 80 % inhibition after 30 min contact time. 12 Precision In a national interlaboratory trial, carried out during summer 1991 by 22 laboratories, precision data were determined. The results are summarized in Annex C. 13 Test report The test report shall refer to this part of ISO
45、11348. The documentation should contain the following information: a) identity of the water sample, including sampling, storage time and conditions; b) c) date of test performance; d) sample pretreatment, if any, e.g. pH after adjustment; e) origin of the bacteria, batch number; date of delivery and
46、 expiration date; f) storage temperature of the bacteria; g) expression of the results in accordance with Clause 10 and Table 1; h) any deviation from this method and information on all circumstances which might affect the results; i) test results with reference substances for the batch of bacteria
47、and the actual test. DIN EN ISO 11348-2:2009-05 EN ISO 11348-2:2008 (E) 14pH and oxygen concentration, in mg/l or % saturation of the original water sample N2); N2) National footnote: Amendment: Prior to test batch. Annex A (informative) Colour-correction method A.1 Application Loss of light due to
48、absorption can occur when a sample shows a visible colour in the dilution series, especially in the red to brown colour range. If there is a visible colour at the EC20concentration, the following procedure is performed to check if colour correction is needed. In any case, when the test sample concen
49、tration is close to the EC50-value, a colour correction should be made. A.2 Additional apparatus A.2.1 Colour-correction tube, double-walled tube, fitting the lumino-meter. A.2.2 Pasteur pipettes. A.3 Procedure Carry out the complete colour-correction procedure at a temperature of 15 C 1C in a thermostatically controlled incubator. Prepare a dilution of the test sample with diluent (5.2) with a concentration close to the EC20,t-value (Ck). When the EC20,t-values differ
