SIST EN ISO 8692:2005

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Wasserbeschaffenheit - Süßwasseralgen-Wachstumshemmtest mit einzelligen Grünalgen (ISO 8692:2004)Qualité de l'eau - Essai d'inhibition de la croissance des algues d'eau douce avec des algues vertes unicellulaires (ISO 8692:2004)Water quality - Freshwater algal growth inhibition test with unicellular green algae (ISO 8692:2004)13.060.70Preiskava bioloških lastnosti vodeExamination of biological properties of waterICS:Ta slovenski standard je istoveten z:EN ISO 8692:2004SIST EN ISO 8692:2005en,fr,de01-februar-2005SIST EN ISO 8692:2005SLOVENSKI
STANDARDSIST EN 28692:19981DGRPHãþD



SIST EN ISO 8692:2005



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN ISO 8692
October 2004 ICS 13.060.70
Supersedes EN 28692:1993
English version
Water quality - Freshwater algal growth inhibition test with unicellular green algae (ISO 8692:2004)
Qualité de l'eau - Essai d'inhibition de la croissance des algues d'eau douce avec des algues vertes unicellulaires (ISO 8692:2004)
Wasserbeschaffenheit - Süßwasseralgen-Wachstumshemmtest mit einzelligen Grünalgen (ISO 8692:2004) This European Standard was approved by CEN on 13 September 2004.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
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B-1050 Brussels © 2004 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 8692:2004: ESIST EN ISO 8692:2005



EN ISO 8692:2004 (E)
2
Foreword
This document (EN ISO 8692:2004) has been prepared by Technical Committee ISO/TC 147 "Water quality" in collaboration with Technical Committee CEN/TC 230 "Water analysis", the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by April 2005, and conflicting national standards shall be withdrawn at the latest by April 2005.
This document supersedes EN 28692:1993.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Endorsement notice
The text of ISO 8692:2004 has been approved by CEN as EN ISO 8692:2004 without any modifications.
SIST EN ISO 8692:2005



Reference numberISO 8692:2004(E)© ISO 2004
INTERNATIONAL STANDARD ISO8692Second edition2004-10-01Water quality — Freshwater algal growth inhibition test with unicellular green algae Qualité de l'eau — Essai d'inhibition de la croissance des algues d'eau douce avec des algues vertes unicellulaires
SIST EN ISO 8692:2005



ISO 8692:2004(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
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SIST EN ISO 8692:2005



ISO 8692:2004(E) © ISO 2004 – All rights reserved iii Contents Page Foreword.iv 1 Scope.1 2 Normative references.1 3 Terms and definitions.1 4 Principle.2 5 Reagents and media.2 6 Apparatus.4 7 Procedure.5 8 Validity criteria.7 9 Calculation.8 10 Expression of results.9 11 Interpretation of results.9 12 Precision.9 13 Test report.10 Annex A (informative)
Rapid screening of wastewater algal growth inhibition.12 Bibliography.15
SIST EN ISO 8692:2005



ISO 8692:2004(E) iv © ISO 2004 – All rights reserved Foreword ISO (the International Organization 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 established 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. 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. Publication 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 patent 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.
SIST EN ISO 8692:2005



INTERNATIONAL STANDARD ISO 8692:2004(E) © ISO 2004 – All rights reserved 1 Water quality — Freshwater algal growth inhibition 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 establish 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 wastewater. 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 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 the 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 definitions 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 µ=proportional rate of increase in cell density per unit of time: 1ddxxtµ= SIST EN ISO 8692:2005



ISO 8692:2004(E) 2 © ISO 2004 – All rights reserved 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 (day−1). 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 test 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 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 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 inoculum 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 conditions. 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 subspicatus Chodat. SIST EN ISO 8692:2005



ISO 8692:2004(E) © ISO 2004 – All 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: Collection of Algal Cultures Inst. Plant Physiology University of Göttingen Nikolausberger Weg 18 D-37073 Göttingen 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 Ambleside Cumbria LA22 OLP United Kingdom Algothèque du laboratoire de cryptogamie Muséum National d'Histoire 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 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 viability[1]. The algae can be easily liberated from the algal beads when needed to perform the toxicity tests. 5.2 Water, deionized or of equivalent purity (conductivity < 10 µS/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 preparation 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 given 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 microbiotests@skynet.be, are an example 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. SIST EN ISO 8692:2005



ISO 8692:2004(E) 4 © ISO 2004 – All rights reserved 5.3 Nutrients. Prepare 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 chemicals used shall be of reagent grade quality. Sterilize the stock solutions by membrane filtration (mean pore diameter 0,2 µm) 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 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 MgCl2⋅6H2O CaCl2⋅2H2O MgSO4⋅7H2O KH2PO4 1,5 g/l 1,2 g/l 1,8 g/l 1,5 g/l 0,16 g/l 15 mg/l 12 mg/l 18 mg/l 15 mg/l 1,6 mg/l Stock solution 2: Fe-EDTA FeCl3⋅6H2O Na2EDTA⋅2H2O 64 mg/l 100 mg/l 64 µg/l 100 µg/l Stock solution 3: trace elements H3BO3a
MnCl2⋅4H2O ZnCl2 CoCl2⋅6H2O CuCl2⋅2H2O Na2MoO4⋅2H2O 185 mg/l 415 mg/l 3 mg/l 1,5 mg/l 0,01 mg/l 7 mg/l 185 µg/l 415 µg/l 3 µg/l 1,5 µg/l 0,01 µg/l 7 µg/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 Temperature-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. SIST EN ISO 8692:2005



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 chamber. Alternatively the algal densities may be determined by an indirect procedure using for instance a fluorimeter (e.g. in vitro fluorescence[2] or DCMU5)-enhanced in vivo fluorescence[3]), when sufficiently sensitive and if shown to be sufficiently well correlated with cell density. The apparatus used 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 and coloured substances at low cell densities. 6.3 Culture flasks, for example 250 ml conical flasks with air permeable stoppers. 6.4 Apparatus for membrane filtration, using filters of mean pore diameter 0,2 µm. 6.5 Autoclave. 6.6 pH meter. 7 Procedure 7.1 Preparation of growth medium Prepare a growth medium by adding an appropriate volume of the nutrient stock solutions (5.3) to water. Add to approximately 500 ml of water: =10 ml of stock solution 1 (5.3); =1 ml of stock solution 2 (5.3); =1 ml of stock solution 3 (5.3); =1 ml of stock solution 4 (5.3). Make up to 1 000 ml with water. When autoclaving is necessary, stock solution 4 should be added afterwards. Before use, equilibrate it by leaving overnight in contact with air, or by bubbling filtered air through it for 30 min. After equilibration, adjust the pH if necessary to 8,1 ± 0,2, using either 1 mol/l hydrochloric acid or 1 mol/l sodium hydroxide solution. This growth medium is buffered by bicarbonate and atmospheric CO2. Different pH values may be obtained by modifying the concentration of HCO3 and/or the atmospheric CO2 concentration (requires closed vessels) as specified in ISO 14442. Should such modifications be required in order to perform a test at a different, specific pH value, these should be clearly motivated and reported. 7.2 Preparation of pre-culture and inoculum A pre-culture shall be started two to four days before the beginning of the test. Growth medium (7.1) is inoculated at a sufficiently low cell density (e.g. 5 × 103 cells/ml to 104 cells/ml for three days pre-culturing) in order to maintain exponential growth until test start. The pre-culture shall be incubated under the same conditions as those in the test (7.6).
5) DCMU = dichlorophenyldimethyl urea (CAS No. 330-54-1). SIST EN ISO 8692:2005



ISO 8692:2004(E) 6 © ISO 2004 – All rights reserved This exponentially growing pre-culture is used as an inoculum for the test. Measure the cell density in the pre-culture immediately before use in order to calculate the required inoculum volume. 7.3 Choice of test sample concentrations Algae should be exposed to concentrations of the test sample in a geometric series with a ratio not exceeding 3,2 (e.g. 1,0 mg/l, 1,8 mg/l, 3,2 mg/l, 5,6 mg/l and 10 mg/l). The concentrations should be chosen to obtain at least one inhibition below and one inhibition above the intended ErCx parameter. Additionally, at l
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