1、DEUTSCHE NORM Amil 1999 German standard methods for the examination of water, waste water and sludge Part 37: Determination of the inhibitory effect of water on the growth of Photobacterium phosphoreum (cell multiDlication inhibition test) (L 37) Bio-assays (group L) DIN - 38412-37 ICs 13.060.01 Deu
2、tsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung - Testverfahren mit Wasserorganismen (Gruppe L) -Teil 37: Bestimmung der Hemmwirkung von Wasser auf das Wachstum von Bakterien (Photobacterium phosphoreum - Zellvermehrungs-Hemmtest) (L 37) In keeping with current practice in sta
3、ndards published by the International Organization for Standardization (ISO), a comma has been used throughout as the decimal marker. Foreword This standard has been prepared by the Normenausschu Wasserwesen (Water Practice Standards Com- mittee) jointly with Study Group Wasserchemie (Water Chemistr
4、y) of the Gesellschaft Deutscher Chemiker (German Chemists Society) (cf. Explanatory notes). Expert assistance and specialized laboratories will be required to perform the analysis specified in this standard. When using the standard, a check shall be made in each individual case as to whether and to
5、 what extent additional boundary conditions have to be specified. Introduction The test organism used in this method is Photobacterium phosphoreum, a halophilic marine bacterium which is a gram-negative, rod-shaped facultative anaerobe with polar flagella belonging to the species Vibrionaceae (Eu ba
6、cteria). 1 Scope The method described in this standard serves to determine the toxicity of water. 2 Normative references This standard incorporates, by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text, and the
7、titles of the publications are listed below. For dated references, subsequent amendments to or revisions of any of these publications apply to this standard only when incorporated in it by amendment or revision. For undated references, the latest edition of the publication referred to applies. DIN 1
8、2036 Narrow mouth, flat bottom flasks with conical ground socket and stopper for laboratory use DIN 12380 Narrow mouth conical flasks for laboratory use DIN 12664-1 One-mark volumetric flasks with flanged rim, conical socket and conical joint, for laboratory use DIN 12680-2 Graduated cylinders with
9、ring marks for laboratory use DIN 12697 Class AS fast delivery graduated pipettes with a waiting time of 15 seconds, for laboratory use DIN 12775 Laboratory thermometers with 0,l “C, 0,2 “C and 0,5 “C scale intervals Continued on pages 2 to 8. Translation by DIN-Sprachendienst. In case of doubt, the
10、 German-language original should be consulted as the authoritative text. No pari of this translation may be reproduced without the prior permission of Ref. No. DIN 38412-37 : 1999-0 _. - V Deutsches Institut fur Normung e VI Berlin uth Verlag GmbH, D-10772 Berlin, hac the exclusive right of sale for
11、 German Standards (DIN-Normen) English price group 06 . Sales No. 0106 02.00 Page 2 DIN 3841 2-37 : 1999-04 DIN 58945-1 Incubators for microbiological purposes - Concepts, requirements and use DIN EN 285 Steam sterilizers - Large sterilizers DIN EN 27027 Water quality - Determination of turbidity (I
12、S0 7027 : 1990) l Bergeys Manual of Systematic Bacteriology, Vol. 1; Krieg, N.R. and Holt, J.G. (eds). Family II: Vibrionaceae. Baltimore, London: Williams b) 2,5 g/i of magnesium sulfate heptahydrate, MgSO, . 7 H,O; c) 5 mVi of glycerol, C3H803; d) 1 g/i of yeast extract; e) 1 O g/i of tryptic case
13、in peptone; f) 20 g/i of high purity agar. Adjust the pH value to (7,2 I0,2) using hydrochloric acid or sodium hydroxide solution. 9.5 Culture media NOTE: Reducing the concentration of nutrient solution may increase the sensitivity to heavy metals. 9.5.1 a) 20 g/i of sodium chloride, NaCi; b) 0,2 g/
14、i of magnesium sulfate heptahydrate, MgSO, . 7 H,O; c) 3 mVi of glycerol, C3H803; d) 5 g/i of tryptic casein peptone; e) 0,5 g/i of yeast extract. Culture medium (for preculture and inoculum) made up of Adjust the pH value to (7,2 I0,2) using hydrochloric acid or sodium hydroxide solution. 9.5.2 Cul
15、ture medium (for test solution) made up of a) 20 g/i of sodium chloride, NaCi; b) 1 ,O g/i of magnesium sulfate heptahydrate, MgSO, . 7 H,O; c) 15 mVi of glycerol, C3H803; d) 25 g/i of tryptic casein peptone; e) 2,5 g/i of yeast extract. Adjust the pH value to (7,2 I0,2) using hydrochloric acid or s
16、odium hydroxide solution. 9.5.3 Dilution water, prepared by dissolving 20 g of sodium chloride in 1 i of water. NOTE: Up to 30 g/i salination may in some cases reduce growth due to sample-induced contamination. 9.6 Preparation of stock cultures Streak the test organism on culture medium (as in subcl
17、ause 9.4) plates and incubate the inoculated Petri dishes for one to seven days at (20 12) “C. Examine the bacterial colonies daily for growth and luminescence, and as soon as luminescent colonies can be detected, transfer them with inoculation loops to fresh plates. Incubate the freshly inoculated
18、Petri dishes for one to seven days at (20 12) “C. Check the plates for growth and luminescence of the bacterial colonies and store at O “C to 5 “C. Transinoculate the stock cultures to fresh culture medium as in subclause 9.4 not more than one month later. 1 O Sampling and sample preparation If poss
19、ible, examine the water samples on the day they are collected. If not, refrigerate them at 2 “C to 4 “C for up to two days or deep-freeze them at about -1 8 “C for up to two weeks. Homogenize the sample by shaking and, if necessary, leave to settle for one hour. After any settling has taken place, a
20、djust the pH value of the supernatant liquor to (7,O I0,2) with hydrochloric acid or sodium hydroxide solution, ensuring that this changes the concentrations of the substances in the water sample as little as possible. Page 5 DIN 3841 2-37 : 1999-04 Salinate the neutralized sample immediately prior
21、to preparing the test solutions by adding 20 g of sodium chloride per litre of sample; if necessary, make up a dilution series using the dilution water. 11 Procedure 11 .I Preculture and inoculum Transfer a sufficient quantity of bacteria (e.g. two to four inoculation loops) from luminescent colonie
22、s of the stock culture to a 250 mi volumetric flask containing 50 mi of culture medium as in subclause 9.5.1. Seal the culture vessels in an air-permeable and sterile manner and incubate them for (1 7 I 1) h at (20 12) “C. Keep the bacteria in suspension (e.g. using a mechanical shaker), and prevent
23、 deposition on the vessel walls. After incubation, dilute the bacterial suspension with culture medium as in subclause 9.5.1 so as to produce a calculated turbidity of 1 O0 FAU. NOTE: Bear in mind that a high turbidity alters the correlation between the dilution factor and absorbance. 11.2 Test solu
24、tion Combine suitable volumes of culture medium as in subclause 9.5.2, test substance, dilution water and inoculum so as to produce check solutions, and test solutions having the desired dilution factor. EXAMPLE 1: Final volume Test substance Culture medium (as in subclause 9.5.2) Dilution water Ino
25、culum EXAMPLE 2: Final volume Test substance Culture medium (as in subclause 9.5.2) Dilution water Inoculum 100 mi 50 mi 20 mi 20 mi 10 ml 200 pl 100 pl 40 pl 40 pl 20 pl The test solution may also be prepared in microtitre plates (as in example 2), provided due regard is paid to the adsorption prop
26、erties of the active ingredient. When examining bodies of water, the concentration of test substance in the test solution shall not exceed 70 YO. Not less than two check solutions, and for every dilution factor two test solutions, as well as a suitable number of blank solutions, shall be examined. I
27、f a statistical validation of the significance is required, at least seven check solutions shall be prepared. 11.3 Incubation Incubate the test, check and blank solutions at (20 12) “C. Keep the bacteria in suspension (e.g. using a mechanical shaker), and avoid deposition on the vessel walls. 11.4 M
28、easurement Determine the turbidity values or their equivalents, with water as the reference, of all the test, check and blank solutions after (7 Il) h. NOTE: If it is not possible to eliminate the effect of any change in colour during incubation due to reaction with the test substance by measuring a
29、t another wavelength, the absorbance of a clear, filtered test solution (using a filter of 0,25 pm pore size) of the appropriate dilution factor may be substituted for the blank value as reference value. 12 Assessment criterion The test results shall be regarded as acceptable if the initial turbidit
30、y of the check solution of 10 FAU has increased by a factor of at least ten during the test period. NOTE: An increase of the FAU value by afactor of ten means that this value has been doubled three to four times. 13 Evaluation Tabulate the turbidity (or equivalent values) measured after the test per
31、iod has elapsed as a function of the dilution ratio, as shown in table 1. Then, calculate the inhibitory effect, H, as a percentage, using the following equation: Page 6 DIN 3841 2-37 : 1999-04 where BK is the biomass (measured value) in the check solution minus the blank value; Bn is the biomass fo
32、rmed in the n-th dilution of the test substance minus the blank value. Table 1: Evaluation Dilution ratio 1 : 1,43 1: 2 1: 3 1: 4 1: 6 1: 8 1 :12 1 :I6 1 :24 1 :32 FAU vali Mean FI Number Turbidity, FAU at 436 nm Blank solutions Test solutions ?s at 436 nm for check solutions: 555; 566; 565; 560; 57
33、9; 565; 578. J of check solutions: 567. if solutions used to determine blank value for the check solution: 5. NOTE: The dilution factors may be altered to meet requirements imposed by the objective. 14 Expression of results In a dilution series containing a decreasing proportion of test substance, t
34、he dilution factor shall be reported at which the inhibitory effect is below 20 YO for the first time (GLw value). 15 Test report The test report shall refer to this method and contain the following details: a) origin of sample, date, time of day and duration of sampling, pH value of the original wa
35、ter sample; b) sample pretreatment, if any (e.g. homogenization, sedimentation time, storage conditions, pH value adjustment); c) test organisms used (type, strain number, origin); d) date of testing; e) test conditions (e.g. incubation conditions: time, temperature, test volume (microtitre plate, t
36、est vessel) and, if appropriate, pH value at the end of the test); f) method of measurement (e.g. determination of turbidity, with specification of wavelength); g) information on the evaluation as in clause 13 and expression of the result as in clause 14 for every dilution factor (GLw value); Page 7
37、 DIN 3841 2-37 : 1999-04 h) other effects (e.g. stimulation of cell multiplication); i) any deviations from this method and an indication of any circumstances that may have affected the results (e.g. solubility, volatility). 16 Results of interlaboratory testing An interlaboratory test carried out i
38、n 1997 yielded the results given in table 2. Table 2: Results of interlaboratory testing Key to symbols: L number of laboratories X overall mean N number of measurements SR reproducibility standard deviation NAP percentage of outliers VR reproducibility coefficient of variation Other relevant standa
39、rds DIN 3841 2-1 DIN 3841 2-34 German standard methods for the examination of water, waste water and sludge - Bio-assays (group L)-General guidelinesforthe design, performance and evaluation of biological tests (L 1) German standard methods for the examination of water, waste water and sludge - Bio-
40、assays (group L) - Determination of the inhibitory effect of waste water on the light emission of Photobacterium phosphoreum (test using preserved luminescent bacteria) (L 34) German standard methods for the examination of water, waste water and sludge - Bio-assays (group L) - Determination of the i
41、nhibitory effect of waste water on the light emission of Photobacterium phosphoreum (L 34) - Preparation of test suspensions (L 341) DIN 3841 2-341 Explanatory notes It is intended to include a note in the revised edition of DIN 38412-1 specifying that the effective concentration, the lethal concent
42、ration and the dilution factor established under given laboratory conditions are toxicological parameters which, although indicative of a possible hazard to water organisms, do not permit any immediate predictions to be made as to how the environment may be affected. This standard incorporates the G
43、erman standard method Determination of the inhibitory effect of waste water on the growth of Photobacterium phosphoreum (cell multiplication inhibition test) (L 37). Standard methods published as DIN Standards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The stan
44、dard methods included in the loose-leaf publication entitled Deutsche finheits- verfahren zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published by Wiley-VCH Verlag and Beuth Verlag GmbH. The standard methods relating to the Wasserhaushaltsgesetz (German Water Management Act) a
45、nd the Abwasserabgabengesetz (German Law on the charges leviable for the discharge of effluent into water), and all the regulations on waste water that have been issued to date are included in DIN- Taschenbuch (DIN Handbook) 230. Standard methods or draft standards bearing the group title German sta
46、ndard methods for the examination of water, waste water and sludge are classified under the following categories (main titles): General information (group A) (DIN 38402) Sensory analysis (group B) (DIN 38403) Physical and physicochemical parameters (group C) (DIN 38404) Anions (group D) (DIN 38405)
47、Cations (group E) (DIN 38406) Page 8 DIN 3841 2-37 : 1999-04 Substance group analysis (group F) (DIN 38407) Gaseous constituents (group G) (DIN 38408) Parameters characterizing effects and substances (group H) (DIN 38409) Biological-ecological methods of analysis (group M) (DIN 3841 O) Microbiologic
48、al methods (group K) (DIN 3841 1) Bio-assays (group L) (DIN 38412) Individual constituents (group P) (DIN 38413) Sludge and sediments (group S) (DIN 38414) Bio-assays with microorganisms (group T) (DIN 38415) In addition to the methods described in the DIN 38402 to DIN 38415 series of standards, the
49、re are a number of European Standards available, which also form part of the collection of German standard methods. Information on Parts of these series of standards that have already been published can be obtained from the offices of the Normenausschu Wasserwesen, telephone (+ 49 30 2601 -2423), or from Beuth Verlag GmbH, Burggrafenstrae 6, D-10787 Berlin.
