ISO INTERNATIONAL STANDARD 21115 First edition 2019-04 Water quality - Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1) Qualitede I'eau-Détermination dela toxicite aigue d'echantillons d'eau et de produits chimiques vis-d-vis de la lignée cellulaire de branchies de poissons (RTgill-W1) Referencenumber IS021115:2019(E) ISO @IS02019 IS021115:2019(E) COPYRIGHTPROTECTEDDOCUMENT @ISO2019 All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either IsO at the address below orIso's memberbody in the countryof therequester. ISO copyright office CP 401 : Ch. de Blandonnet 8 CH-1214 Vernier, Geneva Phone:+41227490111 Fax:+41 22 749 09 47 Email: [email protected] Website: www.iso.org Published in Switzerland ii @ IS0 2019- All rights reserved IS0 21115:2019(E) Contents Page Foreword ..iv Introduction.. ..V 1 Scope.. 2 Normative references 3 Terms and definitions 4 Principle ..2 4.1 Cell viability assay .2 4.2 Keydifferencesinwatersampleandchemicaltestingprocedure .3 5 Interferences .3 5.1 Matrixeffectsbyeffluentsamples .3 5.2 Interferences ofwater constituents or chemicalswithfluorescentdye assays 6 Reagents 6.1 General .4 6.2 Ready-for-use purchased reagents 6.3 Freshly prepared solutions. 6 7 Apparatus and material ..8 7.1 General equipment. 7.2 Whole-water sample/ex preparation .9 7.3 Cell seeding and plate dosing .9 7.4 Preparation/dosing of stock solutions. .9 7.5 Sampling for chemical analysis .10 7.6 Detectionof cytotoxicity ..10 8 Procedure .10 8.1 Cell line used .10 8.2 Seeding cells into 24-well plates .10 8.2.1 General .10 8.2.2 Preparationofworkingmaterialsand solutionsforcell seeding ..10 8.2.3 Seeding cells: step-by-step performance. .11 8.3 Cell exposure .12 8.3.1 General .12 8.3.2 Exposure of cells to water samples .13 8.3.3 Exposure of cells to chemicals. .18 8.4 Determination of cytotoxicity. .21 8.4.1 General. 21 8.4.2 Visual control of cell damage. 22 8.4.3 Preparation of working materials and solutions for cytotoxicity measurement.23 8.4.4 Measuring cytotoxicity-Step-by-step performance 23 8.4.5 Parametersforthefluorescencemeasurement .24 8.4.6 Chemicalanalyses of thetest samples ..24 9 Preparationandexpressionofresults ..24 10 Validity criteria of the test .25 10.1 Nocells-controlwells 25 10.2 Positive control in L-15/ex. .25 10.3 Positive control in w-ws/ex .26 10.4 Solventcontrol .26 Annex A (informative) Routine cell culture of RTgill-W1 ..27 AnnexB (informative)Performance oftheRTgill-W1 cell lineassay .32 Bibliography .38 @ IS0 2019-All rights reserved iii