1、June 2013 Translation by DIN-Sprachendienst.English price group 12No part of this translation 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 1
2、3.060.70!%1w“2021484www.din.deDDIN EN ISO 10710Water quality Growth inhibition test with the marine and brackish water macroalgaCeramium tenuicorne (ISO 10710:2010);English version EN ISO 10710:2013,English translation of DIN EN ISO 10710:2013-06Wasserbeschaffenheit Bestimmung der Wachstumshemmung a
3、uf die marine und stuarine MakroalgeCeramium tenuicorne (ISO 10710:2010);Englische Fassung EN ISO 10710:2013,Englische bersetzung von DIN EN ISO 10710:2013-06Qualit de leau Essai dinhibition de croissance sur la macro algue deaux marine et saumtre Ceramiumtenuicorne (ISO 10710:2010);Version anglaise
4、 EN ISO 10710:2013,Traduction anglaise de DIN EN ISO 10710:2013-06www.beuth.deDocument comprises 25 pagesIn case of doubt, the German-language original shall be considered authoritative.07.13 DIN EN ISO 10710:2013-06 2 A comma is used as the decimal marker. National foreword The text of ISO 10710:20
5、10 has been prepared by Technical Committee ISO/TC 147 “Water quality” and has been taken over as EN ISO 10710:2013 by Technical Committee CEN/TC 230 “Water analysis” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Pract
6、ice Standards Committee), Working Committee NA 119-01-03-05-03 AK Marine Biotests of NA 119-01-03 AA Wasseruntersuchung. Designation of the method: Growth inhibitation test with the marine and brackish water macroalga (Ceramium tenuicorne) (L 56): Method DIN EN ISO 10710 L 56 This standard has been
7、published to implement the Water Framework Directive (WFD), Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy*). The DIN Standards corresponding to the International Standards referred to i
8、n this document are as follows: ISO 1042 DIN EN ISO 1042 ISO 5667-16 DIN EN ISO 5667-16 ISO 8692 DIN EN ISO 8692 ISO 20079 DIN EN ISO 20079 *)Registered in the DITR database of DIN Software GmbH, obtainable from: Beuth Verlag GmbH, 10772 Berlin. DIN EN ISO 10710:2013-06 3 Expert assistance and speci
9、alized laboratories will be required to perform the analyses described in this standard. Existing safety requirements are to be observed. Depending on the objective of the analysis, a check shall be made on a case-by-case basis as to whether and to what extent additional conditions will have to be s
10、pecified. This standard has been prepared by the Normenausschuss Wasserwesen (Water Practice Standards Committee) in collaboration with the Wasserchemische Gesellschaft Fachgruppe in der Gesellschaft Deutscher Chemiker (Water Chemistry Society Division of the German Chemical Society). It is part of
11、the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung (German standard methods for the examination of water, waste water and sludge): Growth inhibitation test with the marine and brackish water macroalga (Ceramium tenuicorne) (L 56). Standard methods published as DIN S
12、tandards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The standard methods included in the loose-leaf publication entitled Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published by Wiley-VCH Verlag GmbH b) C. tenui
13、corne Ktzing Waern (20S and 30S clone originating from the Oslo fjord). This alga is a widely distributed macroalgae species (phylum Rhodophyta) in estuarine and coastal areas. The strains recommended are available in unialgal, non-axenic cultures1). NOTE 1 This growth inhibition test is based on tw
14、o clones, which were formerly regarded as two different species. These species were the marine Ceramium strictum Harvey sensu Kylin and the brackish water species C. tenuicorne Ktzing Waern. Complete interfertility (References 1112) and DNA data (Reference 10) have shown that the two entities belong
15、 to the same species, with C. tenuicorne as the valid name. The marine clone (former C. strictum) used in this test was isolated in 1973 and originates from the Oslo fjord (20S to 25S). It has been maintained as a laboratory culture for over 30 years. The brackish water clone was isolated in 1995 an
16、d originates from the Baltic Sea, 20 km south of the Ask laboratory in northern Baltic proper (6S to 7S). Cultures can be maintained in the medium specified in Clause 5. Regular subculturing is necessary. NOTE 2 Among the red algae, changes occur between haploid and diploid generations. In the growt
17、h inhibition test, the female gametophytic generation is used since it has an even dichotomous growth pattern and the fastest growth rate. In nature it is difficult to distinguish between male and female plants. This can be done in the laboratory where spermatangia are found on the branches of the m
18、ale plants and trichogynes can be seen on the tips of the claws on the female plants. NOTE 3 The Baltic Sea clone can be adapted and used in tests in salinities between 4S and 12S. The marine clone can be used as test organism in salinities between 12S and 32S. 4.2 Natural and artificial seawater 4.
19、2.1 General Natural seawater is used for the cultivation of the algae and either natural or artificial seawater should be used for testing. The type of seawater to be used depends on the objective of the test. When natural seawater is used, care shall be taken to ensure that it is not polluted. Take
20、 special care to avoid contamination of the water by inorganic or organic substances during preparation and storage. Equipment made of copper shall not be used. 4.2.2 Artificial seawater Prepare the stock solutions for artificial seawater according to Table 1. Start with about one third of the desir
21、ed volume of water, add the weighed quantities of chemicals in accordance with Table 1 and make up to volume with water. This stock solution with a salinity of 100S (equivalent to 10 % mass per volume) has a durability of at least six months, if stored in darkness at room temperature. Before use, th
22、e stock solution should be diluted with water to the desired salinity. Adjust the pH to 8,0 0,2 with 1 mol/l HCl or 1 mol/l NaOH. The artificial seawater shall be sterilized by autoclaving or sterile filtration (pore size, 0,2 m) before use. Re-check the pH after sterilization, and adjust if necessa
23、ry to 8,0 0,2 with 1 mol/l HCl or 1 mol/l NaOH, before use. 1) Suitable suppliers are: a) ITM, Department of Applied Environmental Research of Science, Stockholm University, S-106 91 Stockholm, Sweden; b) University of Oslo, Department of Biology, P.O. Box 1066 Blindern, N-0316 Oslo, Norway. This in
24、formation is given for the convenience of users of this document and does not constitute an endorsement by ISO of these suppliers. DIN EN ISO 10710:2013-06 EN ISO 10710:2013 (E) 7 Table 1 Artificial seawater with a salinity of 10 % mass per volume or 100S (adapted from Reference 13) Quantity per 1 l
25、 medium Quantity per 5 l medium Quantity per 10 l medium Substance g/l g/5 l g/10 l NaCl 70,1 351 702 Na2SO411,7 58,7 117 KCl 2,03 10,2 20,3 KBr 0,293 1,47 2,93 Na2B4O710H2O 0,113 0,567 1,13 MgCl26H2O 31,7 158 317 CaCl26H2O 6,6 33 66 SrCl26H2O 0,066 0,334 0,668 4.2.3 Natural seawater Natural seawate
26、r shall be collected from an uncontaminated site. Filter to remove larger particles. Dilute as necessary with water. Salinity should be increased by addition of natural seawater of a higher salinity or with artificial seawater (see Table 1). Check the pH and adjust if necessary to 8,0 0,2 with 1 mol
27、/l HCl or 1 mol/l NaOH. The natural seawater shall be sterilized by autoclaving or sterile filtration (pore size, 0,2 m) before use. Re-check the pH after sterilization, and adjust if necessary to 8,0 0,2 with 1 mol/l HCl or 1 mol/l NaOH, before use. NOTE 1 A paper filter of around 30 m mesh size is
28、 sufficient. NOTE 2 Natural seawater can be stored frozen at temperatures below 18 C for several years before use. 4.3 Nutrients Prepare the six nutrient solutions in water, with the compositions given in Table 2. Solutions 1, 2, 3, 4 and 6 in Table 2 are prepared in 100 ml one-mark volumetric flask
29、s (6.14). A 1 l one-mark volumetric flask (6.14) is recommended for the preparation of stock solution 5, due to the low masses of the trace element reagents. Precipitation in the trace element solution is avoided by adjustment with NaOH to pH 8. The trace element stock solution (solution 5) shall be
30、 diluted 10 times before use in the cultivation media (see Table 3). After this dilution, a freshly prepared iron solution 3 may be added to trace element solution 5 to increase the durability of the iron. The iron solution shall not be older than one month. These stock solutions will eventually be
31、diluted according to Table 3 to obtain the final nutrient concentrations in the growth and test media. The final concentrations in the media are given in the two rightmost columns of Table 2. 4.4 Media Additions of stock solutions to salt water for preparation of media are shown in Table 3. For cult
32、ivation, testing in natural seawater, and testing in artificial seawater, additions shall be made in accordance with columns A, B, and C, respectively. DIN EN ISO 10710:2013-06 EN ISO 10710:2013 (E) 8 Table 2 Nutrient stock solutions (adapted from Reference 9) Levels in the medium after additions ac
33、cording to Table 3 as elemental mass concentration elemental amount of substance concentration Reagent Compound mass concentration g/l mol/l 1 Nitrogen solution KNO35 000 mg/100 ml 3 462 (N) 247 (N) 2 Phosphorus solution KH2PO4680 mg/100 ml 775 (P) 25 (P) 3 Iron solution FeCl36H2O 100 mg/100 ml 103
34、(Fe) 1,9 (Fe) 4 Carbon solution NaHCO35 760 mg/100 ml 16 480 (C) 1 370 (C) 5 Trace element solution Na2EDTA 6 000 mg/l MnSO4H2O 620 mg/l 10 (Mn) 0,18 (Mn) ZnSO47H2O 250 mg/l 2,84 (Zn) 0,043 (Zn) Na2MoO42H2O 130 mg/l 2,58 (Mo) 0,027 (Mo) CoSO47H2O 4 mg/l 0,042 (Co) 0,000 7 (Co) CuSO45H2O 4 mg/l 0,05
35、(Cu) 0,000 8 (Cu) 6 Vitamins Thiamine (B1) 10 mg/100 ml 50 Cyanocobalamin (B12) 0,1 mg/100 ml 0,5 Biotin 0,1 mg/100 ml 0,5 Table 3 Additions of stock nutrient solutions to seawater for preparation of growth and test medium A B C Growth medium in natural seawater for cultivation Test medium in natura
36、l seawater Test medium in artificial seawater Stock solutions ml/l ml/l ml/l 1 Nitrogen solution 0,5 0,5 0,5 2 Phosphorus solution 0,5 0,5 0,5 3 Iron solution 0,5 0,5 0,5 4 Carbon solution 2 5 Trace element solution diluted 10 times 0,5 6 Vitamin solution 0,5 DIN EN ISO 10710:2013-06 EN ISO 10710:20
37、13 (E) 9 Preparation of 1 l of growth medium or test medium is shown in Table 3. For the cultivation of stock cultures, sterile natural seawater shall be used. For the growth inhibition test, either sterile artificial or natural seawater may be used depending on the objective (see Clause 4). 5 Culti
38、vation Stock cultures of female gametophytes of C. tenuicorne are cultivated in natural seawater (see 4.2.3) where nutrients have been added according to Table 3, column A. The brackish water clone is cultivated at a salinity of 7S and the marine clone (former C. strictum) at salinities of 20S and 3
39、0S. The maintenance of the cultures is facilitated by working under aseptical conditions. C. tenuicorne should be grown in sterile plastic or glass Petri dishes (e.g. 90 mm diameter, 15 mm height). The algae are cultured at 22 C 2 C, at a light intensity of 35 mol m2s1 7 mol m2s1and a light regime o
40、f 14 h light and 10 h darkness. These conditions can be achieved on a laboratory bench with an ordinary time-regulated lamp at about 350 mm distance. To maintain actively growing female gametophytes, plants have to be transferred to fresh medium each week. The procedure is to fill sterile Petri dish
41、es with approximately 25 ml sterile culture medium according to Table 3 column A. Approximately 20 to 30 tips of length 10 mm to 20 mm from female plants are transferred to each dish. If the test is intended to be performed at a salinity other than 7S, 20S or 30S, the algae need to be adapted to the
42、 new salinity prior to the start of the test. Seawater of other salinities is prepared according to 4.2.2 and 4.2.3. The algae should be adapted successively by transferring the algae every other day into fresh media with an increase or decrease in salinity of 3S. The algae shall be cultivated for a
43、t least two weeks in the final test salinity prior to the start of the test. NOTE 1 To increase the stability of iron, iron solution 3 can be added to the diluted trace element solution. NOTE 2 Back-up cultures can be held for up to two months without refreshing if cultivated under lower light inten
44、sities at room temperature. If the temperature is at approximately 10 C, the algae can be kept for up to three months. 6 Apparatus All equipment that has contact with the test medium shall be made of glass or other chemically inert material. Usual laboratory equipment and in particular the following
45、. 6.1 Temperature-controlled cabinet or room, with a white fluorescent light providing even illumination, suitable for the lighting requirements specified for the test in 7.5. 6.2 Fluorescent tubes of the daylight or “warm white” type, capable of providing a light intensity of (70 7) mol m2s1for the
46、 exposure period, and around (35 7) mol m2s1for cultivation. 6.3 Timer, to control a light regime of 14 h light and 10 h darkness. 6.4 Light meter, which measures photons or energy (expressed in micromoles per square metre per second), within the photosynthetic range 400 nm to 700 nm, is preferred.
47、6.5 pH meter, for the measurement and adjustment of pH during the preparation of culture and test solutions and to measure pH at the termination of a test. 6.6 Salinity meter or conductivity meter, to measure and adjust the salt content of the culture and dilution water. DIN EN ISO 10710:2013-06 EN
48、ISO 10710:2013 (E) 10 6.7 Stereomicroscope, with a magnification of 6 times to 10 times, for cutting female plants prior to a test and for measuring the length of the plants. 6.8 Sterilization equipment. All equipment and solutions should be sterile to prevent contamination. This can be done in an autoclave. Solutions can also be sterilized by filtration (pore size, 0,2 m). Glassware can be sterilized in a muffle furnace at 150 C for 3 h. 6.9 Gas burne
