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1、 Reference number ISO 8692:2004(E) ISO 2004 INTERNATIONAL STANDARD ISO 8692 Second edition 2004-10-01 Water quality Freshwater algal growth inhibition test with unicellular green algae Qualit de leau Essai dinhibition de la croissance des algues deau douce avec des algues vertes unicellulaires Copyr
2、ight International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 20:59:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 8692:2004(E) PDF disclaimer This PDF file may co
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6、rwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyrigh
7、t office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2004 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Techn
8、ical Standards 1/9972545001 Not for Resale, 04/19/2007 20:59:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 8692:2004(E) ISO 2004 All rights reserved iii Contents Page Forewordiv 1 Scope1 2 Normative references .1 3 Terms and definitions.1 4 Principle.2 5 Reagents a
9、nd media .2 6 Apparatus.4 7 Procedure.5 8 Validity criteria 7 9 Calculation.8 10 Expression of results9 11 Interpretation of results9 12 Precision9 13 Test report10 Annex A (informative) Rapid screening of wastewater algal growth inhibition.12 Bibliography .15 Copyright International Organization fo
10、r Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 20:59:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 8692:2004(E) iv ISO 2004 All rights reserved Foreword ISO (the International Organ
11、ization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been establish
12、ed has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
13、 International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Pu
14、blication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such paten
15、t rights. ISO 8692 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. This second edition cancels and replaces the first edition (ISO 8692:1989), which has been technically revised. Copyright International Organization for Standardization Provided b
16、y IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 20:59:05 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 8692:2004(E) ISO 2004 All rights reserved 1 Water quality Freshwater algal growth inhib
17、ition test with unicellular green algae WARNING Persons using this International Standard should be familiar with normal laboratory practice. This International Standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to e
18、stablish appropriate safety and health practices and to ensure compliance with any national regulatory conditions. 1 Scope This International Standard specifies a method for the determination of the growth inhibition of unicellular green algae by substances and mixtures contained in water or by wast
19、ewater. This method is applicable for substances that are easily soluble in water. With modifications to this method, as described in ISO 14442 and ISO 5667-16, the inhibitory effects of poorly soluble organic and inorganic materials, volatile compounds, heavy metals and waste water can be tested. A
20、 rapid algal growth inhibition screening test for wastewater is included in Annex A. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of t
21、he referenced document (including any amendments) applies. ISO 5667-16:1998, Water Sampling Part 16: Guidance on biotesting of samples ISO 14442:1999, Water quality Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water 3 Terms and defi
22、nitions For the purposes of this document, the following terms and definitions apply. 3.1 cell density x number of cells per unit volume of medium NOTE Cell density is expressed in cells per millilitre. 3.2 specific growth rate proportional rate of increase in cell density per unit of time: 1 d d x
23、xt = Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 20:59:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 8692:2004(E) 2 ISO 2004 All rights res
24、erved where x is the cell density, expressed in cells per millilitre; t is the time, expressed in days NOTE Specific growth rate is expressed in inverse days (day1). 3.3 growth medium mixture of water and nutrients in which algal cells are incubated, which is used for pre-cultures and controls 3.4 t
25、est sample aqueous sample (e.g. wastewater), chemical substance or mixture for which the inhibitory effects on the growth of algae are determined 3.5 test medium mixture of water, nutrients and test sample 3.6 test batch mixture of water, nutrients and test sample (test medium 3.5), inoculated with
26、algae 3.7 control mixture of water, nutrients (growth medium 3.3) without test sample, inoculated with algae 3.8 effective concentration ErCx concentration of test sample which results in a reduction of x % in the specific growth rate relative to the controls NOTE To unambiguously denote an EC value
27、 deriving from growth rate it is proposed to use the symbol “ErC”. 4 Principle Monospecies algal strains are cultured for several generations in a defined medium containing a range of concentrations of the test sample, prepared by mixing appropriate quantities of growth medium, test sample and an in
28、oculum of exponentially growing algal cells. The test batches are incubated for a period of (72 2) h during which the cell density in each test solution is measured at least every 24 h. Inhibition is measured as a reduction in growth rate, relative to control cultures grown under identical condition
29、s. 5 Reagents and media 5.1 Test organism, using either of the following planktonic fresh water algae species: a) Desmodesmus subspicatus1) (86.81 SAG). b) Pseudokirchneriella subcapitata (Korshikov) Hindak2) (ATCC 22662, CCAP 278/4 or 61.81 SAG). 1) This species is formerly known as Scenedesmus sub
30、spicatus Chodat. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 20:59:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 8692:2004(E) ISO 2004 All
31、rights reserved 3 NOTE 1 Both algae species are planktonic green algae belonging to the order of Chlorococcales (Chlorophyta, Chlorophyceae), and are usually unicellular in culture. The strains recommended are available in unialgal, non-axenic cultures from the following collections3). SAG: Collecti
32、on of Algal Cultures Inst. Plant Physiology University of Gttingen Nikolausberger Weg 18 D-37073 Gttingen Germany ATCC: American Type Culture Collection 12301 Parklane Drive Rockville Maryland 20852 USA CCAP: Culture Centre of Algae and Protozoa Freshwater Biological Association The Ferry House Ambl
33、eside Cumbria LA22 OLP United Kingdom Algothque du laboratoire de cryptogamie Musum National dHistoire Naturelle 12, rue Buffon 75005 Paris France NOTE 2 Stock cultures can be maintained in the medium described in 5.3. and 7.1. However, a frequent sub-culturing is necessary (once a week) to prevent
34、failure of growth. The stock culture can be maintained for extended periods on richer algal media such as those recommended by the culture collection. Alternatively algae can be stored for several months in alginate beads4), without losing their viability1. The algae can be easily liberated from the
35、 algal beads when needed to perform the toxicity tests. 5.2 Water, deionized or of equivalent purity (conductivity 10 S/cm), for use in the preparation of the growth medium and test substance solutions. Take special care to avoid contamination of the water by inorganic or organic substances during p
36、reparation and storage. Equipment made of copper shall not be used. 2) This species is formerly known as Selenastrum capricornutum Prinz. The new name is currently cited by culture collections. 3) Trade name of strains are examples of suitable strains available commercially. This information is give
37、n for the convenience of users of this International Standard and does not constitute an endorsement by ISO of these products. 4) The algae beads supplied by MICROBIOTESTS Inc., Venecoweg 19, 9810 Nazareth, Belgium. Tel. (32) 9 380 8545, fax (32) 9 380 8546, e-mail microbiotestsskynet.be, are an exa
38、mple of a suitable commercially available product. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the same results. Copyright Internat
39、ional Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 20:59:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 8692:2004(E) 4 ISO 2004 All rights reserved 5.3 Nutrients. Pr
40、epare four nutrient stock solutions in water, according to the compositions given in Table 1. These solutions are eventually diluted (see 7.1 and 7.4) to achieve the final nutrient concentrations in the test solutions. However, the macro-nutrients may instead be added directly to the water. All chem
41、icals used shall be of reagent grade quality. Sterilize the stock solutions by membrane filtration (mean pore diameter 0,2 m) or by autoclaving (120 C, 15 min). Store the solutions in the dark at 4 C. Do not autoclave stock solution 4 in order to avoid evaporative loss of NaHCO3, but sterilize it by
42、 membrane filtration. Table 1 Mass concentrations of nutrients in the test solution Stock solution Nutrient Mass concentration in stock solution Final mass concentration in test solution Stock solution 1: macro-nutrients NH4Cl MgCl26H2O CaCl22H2O MgSO47H2O KH2PO4 1,5 g/l 1,2 g/l 1,8 g/l 1,5 g/l 0,16
43、 g/l 15 mg/l 12 mg/l 18 mg/l 15 mg/l 1,6 mg/l Stock solution 2: Fe-EDTA FeCl36H2O Na2EDTA2H2O 64 mg/l 100 mg/l 64 g/l 100 g/l Stock solution 3: trace elements H3BO3a MnCl24H2O ZnCl2 CoCl26H2O CuCl22H2O Na2MoO42H2O 185 mg/l 415 mg/l 3 mg/l 1,5 mg/l 0,01 mg/l 7 mg/l 185 g/l 415 g/l 3 g/l 1,5 g/l 0,01
44、g/l 7 g/l Stock solution 4: NaHCO3 NaHCO3 50 g/l 50 mg/l a H3BO3 can be dissolved by the addition of 0,1 mol/l NaOH. 6 Apparatus All equipment that comes in contact with the test medium shall be made of glass or other chemically inert material. Standard laboratory apparatus and the following. 6.1 Te
45、mperature-controlled cabinet or room, with a white fluorescent light, providing continuous, uniform illumination suitable for the lighting requirements as specified for the test in 7.6. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Tech
46、nical Standards 1/9972545001 Not for Resale, 04/19/2007 20:59:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 8692:2004(E) ISO 2004 All rights reserved 5 6.2 Apparatus for measuring algal cell density, preferably a particle counter, or a microscope and a counting cha
47、mber. Alternatively the algal densities may be determined by an indirect procedure using for instance a fluorimeter (e.g. in vitro fluorescence2 or DCMU5)-enhanced in vivo fluorescence3), when sufficiently sensitive and if shown to be sufficiently well correlated with cell density. The apparatus use
48、d shall be capable of measuring cell densities as low as 104 cells/ml and to distinguish between algal growth and disturbing effects, for example the presence of particulate matter and the colour of the sample. Spectrophotometers may be sufficiently sensitive to measure 104 cells/ml providing a sufficient path length (up to 10 cm) can be used. However, this technique is particularly sensitive to interferences from suspended material a