EN 28692:1993

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Kakovost vode - Preskus zaviranja rasti sladkovodnih alg s Scenedesmus subspicatus in Selenastrum capriocornutum (ISO 8692:1989)Wasserbeschaffenheit - Wachstumshemmtest mit den Süßwasseralgen Scenedesmus subspicatus und Selenastrum capricornutum (ISO 8692:1989)Qualité de l'eau - Essai d'inhibition de la croissance des algues d'eau douce avec Scenedesmus subspicatus et Selenastrum capricornutum (ISO 8692:1989)Water quality - Fresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricornutum (ISO 8692:1989)13.060.70Preiskava bioloških lastnosti vodeExamination of biological properties of waterICS:Ta slovenski standard je istoveten z:EN 28692:1993SIST EN 28692:1998en01-januar-1998SIST EN 28692:1998SLOVENSKI
STANDARDSIST ISO 8692:19971DGRPHãþD



SIST EN 28692:1998



SIST EN 28692:1998



SIST EN 28692:1998



INTERNATIONAL STANDARD IS0 8692 First edition 1989-l 1-15 Water quality - Fresh water algal growth inhibition test with Scenedesrnus subspicatus and Selenas trum capricornu turn Qua/it6 de l’eau - Essai d’inhibition de la croissance des Scenedesmus subspicatus et Selenastrum capricornu turn algues d’eau deuce avec Reference number IS0 8692 : 1989 (E) SIST EN 28692:1998



Is0 8692 : 1989 (El Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 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, govern- mental and non-governmental, in liaison with ISO, also take part in the work. IS0 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for approval before their acceptance as International Standards by the IS0 Council. They are approved in accordance with IS0 procedures requiring at least 75 % approval by the member bodies voting. International Standard IS0 8692 was prepared by Technical Committee ISO/TC 147, Water quality, Annex A of this International Standard is for information only. 0 IS0 1989 All rights reserved. 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 the publisher. International Orga nization for Standardization Case postale 560 CH-121 1 Geneve 20 l Switzerland Printed in Switzerland SIST EN 28692:1998



INTERNATIONAL STANDARD IS0 8692 : 1989 (E) Water quality - Fresh water algal growth inhibition test with Scenkdesmus subspia tus and Selenastrum capricornutum 1 Scope This International Standard specifies a method for the deter- mination of the toxic effects of chemical compounds on the growth of planktonic freshwater algae. The test can be used for readily water-soluble substances which are not significantly degraded or eliminated from the test system. 2 Principle Monospecific algal strains are cultured for several generations in a defined medium containing a range of concentrations of the test substance prepared by mixing appropriate quantities of nutrient concentrate, water, test substance stock solutions, and an inoculum of exponentially growing algal cells. The test solutions are incubated for a minimum period of 72 h, during which the cell density in each is measured at least every 24 h. Inhibition is measured as a reduction in growth or growth rate relative to control cultures grown under identical conditions. 3 Definitions and abbreviations For the purposes of this International Standard, the following definitions and abbreviations apply. 3.1 cell density : Number of cells per unit volume. 3.2 growth : Increase in cell density. 3.3 growth rate : Expression of rate of increase in cell dens- ity with respect to time as given in 8.2.2. 3.4 test solution : Mixture of water, nutrients and test substance in which algal cells are incubated. 3.5 control : Mixture of water, nutrients and algal cells without test substance. 36 median effective concentration (E&l : The concen- tration of test substance which results in a 50 % reduction in either growth or growth rate relative to the controls. 3.7 no observed effect concentration (NOEC) : The highest concentration tested at which there is no statistically significant reduction of growth or growth rate relative to the controls. 4 Materials 4.1 Test organism Use either of the following planktonic freshwater algae : a) Scenedesmus subspicatus Chodat (86.81 SAG) or b) Selenastrum capricornutum Printz (ATCC 22662 or CCAP 278/4). ‘) NOTE - Both species are planktonic green algae belonging to the order of Chlorococcales lchlorophyta, Chlorophyceael, and are usually unicellular in culture. The strains recommended are available in unialgal, non-axenic cultures from the following collections : 86.81 SAG : Collection of Algal Cultures Inst. Plant Physiology University of Gijttingen Nikolausberger Weg 18 D-3400 Gottingen Germany, F.R. ATCC22662 : American Type Culture Collection 12301 Parklane Drive Rockville Maryland 20852 USA 1) This species is now systematically named Raphidocell’s subcapitata Korsikov nov. comb. [l]. SIST EN 28692:1998



Is0 8692 : 1989 (El CCAP 27814 : Culture Centre of Algae and Protozoa Freshwater Biological Association The Ferry House Ambleside Cumbria LA22 OLP, UK Algotheque du laboratoire de Cryptogamie Museum d’histoire naturelle 12, rue Buffon F-75665 Paris, France 4.2 Water All water used in the preparation of the nutrient medium and test substance solutions shall be deionized or of equivalent quality. Take special care to avoid contamination of the water by inorganic or organic substances during preparation and storage. No copper equipment shall be used. 4.3 Nutrients Prepare four stock solutions in water, according to the com- positions given in table 1. NOTE - These solutions will eventually be diluted (see 6.1 and 6.4) to achieve the final nutrient concentrations in the test solutions. Table 1 Nutrient Concentration Final concentration in stock solution in test solution Stock solution 1 : macro-nutrients NH&I 1,5 g.I-’ 15 mg.l-1 MgC12m6H20 I,2 g.I-’ 12 mg.l-’ CaCl2.2H20 I,8 g-l-’ 18 mg.l-’ MgS04.7H20 1,5 g-I-1 15 mg.l-’ KH2P04 0,16 g-l-’ I,6 mg.l-’ Stock solution 2 : Fe-EDTA FeCl3.6H20 80 mg.l-’ 80 pg.l-’ Na2EDTAm2H20 100 mg.I-’ 100 ug.I-’ Stock solution 3 : trace elements H3B03 185 mg.l-’ 185 pg.1 - ’ MnCl2*4H20 415 mg.l-’ 415 l.lg.l-’ ZnCI2 3 mggI - ’ 3 l.lg.l-’ CoCl2.6H2O 1,5 mg.l-’ 1,5 ug.l-’ CuC12=2H20 0,Ol rng-I-’ 0,Ol pg.l-’ Na2Mo04.2H20 7 mg.l- ’ 7 pg.l-’ Stock solution 4 : NaHC03 NaHC03 50 g*I-’ 50 mg.l-’ All the chemicals used shall be of reagent grade quality. Sterilize the stock solutions by membrane filtration (mean pore diameter 0,2 pm) or by autoclaving (120 OC, 15 min). Store the solutions in the dark at 4 OC. Do not autoclave stock solution 4 (NaHC03) but sterilize it only by membrane filtration. 5 Apparatus All equipment in contact with the test medium shall be made of glass or chemically inert material. Ordinary laboratory apparatus and 5.1 Temperature-controlled cabinet or room with con- tinuous even illumination by white fluorescent light suitable to meet requirements with respect to lighting conditions during the test as specified in 6.6. 5.2 Apparatus for measuring algal cell density, preferably a particle counter, or microscope with counting chamber. Alternatively determine the state of growth of the algal cultures by an indirect procedure using a spectro- photometer, turbidimeter or fluorimeter when sufficiently sen- sitive and if shown to be sufficiently well correlated with cell density. The apparatus used shall be capable of measuring ac- curately cell densities as low as lo4 cells per millilitre. 5.3 Culture flasks, e.g. 250 ml conical flasks with air- permeable stoppers. 5.4 Apparatus for membrane filtration, using filters of mean pore diameter 0,2 pm. 5.5 Autoclave. 5.6 pH meter. 6 Procedure 6.1 Preparation of nutrient concentrate Prepare a nutrient concentrate as follows (for 1 000 ml) : Add to 100 ml of stock solution 1 (4.3) : 10 ml of stock solution 2 (4.3) 10 ml of stock solution 3 (4.3) and 10 ml of stock solution 4 (4.3). Make up to 1 000 ml with water. Prepare the nutrient concentrate freshly before each test. 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 8,3 + 0,2, using either 1 mol/l hydrochloric acid or 1 mol/l sodium hydroxide solution. 6.2 Preparation of inoculum The algal inoculum for the test shall be taken from an exponen- tially growing pre-culture. Set up the pre-culture 3 days before the start of the test as described below. Mix one part by volume of nutrient concentrate (6.1) with eight parts of water. Add sufficient cells from the algal stock culture so that, when made up to 10 parts with water, the cell density is of the order of lo4 cells per millilitre. 2 SIST EN 28692:1998



IS0 8692 : 1989 (El Maintain the pre-culture under the same conditions as used in the test (see 6.6) for 3 days, after which time it should be in ex- ponential growth and of sufficient cell density to be used as an inoculum for the test. Measure the cell density in the preculture immediately before use (see 6.7), in order to calculate the required inoculum volume. 63 . Choice of test concentrations The concentrations of test substance to be tested shall nor- mally follow a geometric progression, for example 10; 3,2; l,O; 0,32; . . .; 0,Ol mgJ-I. If possible the concentrations shall be chosen to obtain several (4 to 5) levels of effect ranging from < 10 % to > 90 % inhi- bition of growth. NOTE - A suitable concentration range is best determined by carrying out a preliminary range-finding test covering several orders of magnitude of difference in test concent
...