1、BSI Standards PublicationBS ISO 19820:2016Water quality Determination of the acute toxicity to the marine rotifer Brachionus plicatilisBS ISO 19820:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 19820:2016.The UK participation in its preparation was entru
2、sted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British
3、 Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 87413 0ICS 13.060.70Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 March 2016
4、.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 19820:2016 ISO 2016Water quality Determination of the acute toxicity to the marine rotifer Brachionus plicatilisQualit de leau Dtermination de la toxicit aigue envers le rotifre marin Brachionus plicatilisINTERNATIONAL
5、 STANDARDISO19820First edition2016-03-01Reference numberISO 19820:2016(E)BS ISO 19820:2016ISO 19820:2016(E)ii ISO 2016 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or u
6、tilized 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 or ISOs member body in the country of the requester.ISO copyr
7、ight officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 19820:2016ISO 19820:2016(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Test environment . 26 R
8、eagents, test organisms and media . 26.1 Test organisms 26.2 Culturing and dilution medium 26.3 Reference substance . 37 Apparatus . 38 Treatment and preparation of samples 38.1 Special precautions 38.2 Preparation of the stock solutions of substances to be tested . 49 Procedure. 49.1 Selection of t
9、est concentrations 49.2 Preparation of the test and control solutions . 49.3 Introduction of the organisms 59.4 Incubation of the test system 69.5 Measurements 610 Estimation of the LC50. 711 Reference test 712 Validity criteria 713 Test report . 8Annex A (informative) Procedure for hatching of Brac
10、hionus plicatilis cysts . 9Annex B (informative) Preparation of artificial seawater .10Annex C (informative) Performance data .11Bibliography .13 ISO 2016 All rights reserved iiiContents PageBS ISO 19820:2016ISO 19820:2016(E)ForewordISO (the International Organization for Standardization) is a world
11、wide 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 t
12、hat 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.The procedures used to develop this doc
13、ument and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directi
14、ves, Part 2 (see www.iso.org/directives).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. Details of any patent rights identified during the developme
15、nt of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO speci
16、fic terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 147, Water quality,
17、Subcommittee SC 5, Biological methods.iv ISO 2016 All rights reservedBS ISO 19820:2016ISO 19820:2016(E)IntroductionThe evaluation of harmful effects on chemicals and pollutants on the biota in marine and estuarine environments has, for several years, involved the performance of biological tests.Roti
18、fers, and especially the species Brachionus plicatilis, are of interest from the ecotoxicological view because they are often an important component of the zooplankton and serve as prey for small fish and larger invertebrates.The test specified in this International Standard involves determination o
19、f the lethal effects of toxicants to the marine rotifer, Brachionus plicatilis, after 24 h or 48 h exposure (depending on the intended use of the results). Prolongation of the exposure to 48 h is advised since it substantially increases the sensitivity of the assay. ISO 2016 All rights reserved vBS
20、ISO 19820:2016BS ISO 19820:2016Water quality Determination of the acute toxicity to the marine rotifer Brachionus plicatilisWARNING Persons using this document should be familiar with normal laboratory practice. This document does not purport to address all of the safety problems, if any, associated
21、 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.IMPORTANT It is absolutely essential that tests conducted in accordance with this document be carried out by suitably qualified st
22、aff.1 ScopeThis International Standard specifies a method for the determination of the lethal effects of toxicants to Brachionus plicatilis after 24 h or 48 h exposure.The method is applicable to the following:a) chemical substances which are soluble or which can be maintained as a stable suspension
23、 ofdispersions under the conditions of the test;b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration, orcentrifugation;c) marine or estuarine waters;d) sediment elutriates/eluates.2 Normative referencesThe following documents, in whole or in part, are
24、 normatively referenced in this document and are indispensable for its application. 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, Water quality Sampling Part 16: Guidance on
25、biotesting of samplesISO 5814, Water quality Determination of dissolved oxygen Electrochemical probe methodISO 10523, Water quality Determination of pH3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1control batchseries of replicates containing c
26、ontrol solutionSOURCE: ISO 6341:2012, 3.13.2LC50concentration of dilution of the test sample which gives rise to 50 % mortality of the test organismsINTERNATIONAL STANDARD ISO 19820:2016(E) ISO 2016 All rights reserved 1BS ISO 19820:2016ISO 19820:2016(E)3.3test batchseries of replicates filled with
27、the same test solutionSOURCE: ISO 6341:2012, 3.63.4pure waterdeionized or distilled water with a conductivity below 10 S/cm4 PrincipleThe test organisms are exposed to a range of concentrations of the sample under analysis and the percentage mortality of the test organisms is determined after 24 h e
28、xposure and/or 48 h exposure with subsequent calculation of the 24 h LC50and/or the 48 h LC50.Prolongation of the test to 48 h is recommended since it substantially increases the sensitivity of the assay.The test is carried out in one or two of the following stages: a “range-finding test” to determi
29、ne the range of concentrations or dilutions needed for calculation of the 24 h LC50or the 48 h LC50; a “definitive test” conducted when the data of the range-finding test are not sufficient or adequate for calculation of the 24 h LC50or the 48 h LC50.5 Test environmentThe test shall be carried out i
30、n the dark, in a temperature-controlled room, or incubator at (25 1) C in the test containers.Maintain the atmosphere free from toxic dusts or vapours. The use of control solutions is a double check that the test is performed in an atmosphere free from toxic dusts and vapours.6 Reagents, test organi
31、sms and mediaUse only reagents of recognized analytical grade, unless otherwise specified.6.1 Test organismsThe tests organisms are females of the species Brachionus plicatilis obtained from a laboratory culture (see References 5,7, and 8) or hatched from commercially available cysts.1)The procedure
32、 for hatching of Brachionus plicatilis from cysts is described in Annex A.6.2 Culturing and dilution mediumA natural or an artificial seawater may be used as the water for stock culturing the rotifers or as dilution water for the testing. Natural seawater shall be collected from an unpolluted locati
33、on and should have salinity between 29 and 36 . Natural seawater must be filtered (30 m) and conditioned to test temperature and oxygen saturation prior to use. Natural seawater can be stored cold (4 1) C for several weeks.An example of artificial seawater suitable for culturing and testing is given
34、 in Annex B.1) MicroBioTests Inc. Mariakerke, Belgium, is an example of a supplier able to provide suitable Brachionus plicatilis cysts commercially. This information is given for the convenience of the users of this document and does not constitute an endorsement by ISO of this supplier.2 ISO 2016
35、All rights reservedBS ISO 19820:2016ISO 19820:2016(E)6.3 Reference substancePotassium dichromate (K2Cr2O7) or copper sulfate (CuSO45H2O) are recommended as reference chemicals.NOTE Since K2Cr2O7is a carcinogenic substance toxic via inhalation, the use of a ready-made solution with a defined concentr
36、ation of K2Cr2O72)for the preparation of the stock solution of the reference substance can reduce the risk of inhalation of the toxic dust in the laboratory.7 ApparatusUsual laboratory equipment and, in particular, the following.7.1 Temperature-controlled room or chamber.7.2 Petri dishes.Small Petri
37、 dishes (diameter 5 cm) in glass or in inert plastic material.7.3 Test containers.Disposable 48 (6 8) microplates made from chemically inert material.7.4 Pipette for sampling rotifers, with a sufficient diameter for capturing the animals while allowing sampling of only a small volume of medium.For e
38、xample, single use 1 ml capillary mini-pipettes are suitable.7.5 Stereomicroscope with incident (bottom) illumination, with a magnification of at least eight times and, if possible, a continuous magnification.7.6 Light source, providing a range of light intensity in the hatching Petri dish of 3 000
39、lx to 4 000 lx corresponding to 40 mol.m-2.s-1to 55 mol.m-2.s-1.7.7 Sample collecting bottles, as specified in ISO 5667-16.8 Treatment and preparation of samples8.1 Special precautionsSpecial precautions are required for sampling, transportation, storage, and treatment of seawater or effluent.Sampli
40、ng, transportation, and storage of the samples should be performed as specified in ISO 5667-16.Carry out the toxicity test as soon as possible, ideally within 12 h of collection. If this time interval cannot be met, cool the sample to 0 C to 5 C and test the sample within 24 h. If it is not possible
41、 to perform the test within 72 h, the sample may be frozen and maintained deep-frozen (below 18 C) for testing within two months of collection, provided that characteristics are known to be unaffected by freezing. At the time of testing, homogenize the sample to be analysed by shaking manually, and,
42、 if necessary, allow to settle for 2 h in a container and sample by drawing off (using a pipette) the required quantity of supernatant maintaining the end of the pipette in the centre of the section of the test tube and halfway between the surface of the deposited matters and the surface of the liqu
43、id.2) Titrisol potassium dichromate solution is an example of a suitable product available commercially. This information is given for the convenience of the users of this document and does not constitute an endorsement by ISO of this product. ISO 2016 All rights reserved 3BS ISO 19820:2016ISO 19820
44、:2016(E)If the raw sample of the decanted supernatant is likely to interfere with the test (due to the presence of residual suspended matter, protozoa, microorganisms, etc.), filter or centrifuge the raw or decanted sample.The sample obtained by either of these methods is the sample submitted to tes
45、ting.Measure the dissolved oxygen concentration (as specified in ISO 5814) and record the value (mg/l) in the test report.8.2 Preparation of the stock solutions of substances to be testedPrepare the stock solution of the substance to be tested by dissolving a known quantity of substance in a specifi
46、ed volume of test medium (6.2) at the time of use. However, if the stock solution of the substance is stable under certain conditions, it may be prepared in advance and stored under these conditions.For substances sparingly soluble in the test medium, refer to the specifications given in ISO 5667-16
47、.9 Procedure9.1 Selection of test concentrationsThe test should comprise at least five concentrations of the sample to be tested. The dilutions shall be selected within a geometric series with a separation factor which depends on the nature of the sample to be analysed (chemical substances, effluent
48、s, waters) and of the type of assay (range finding or definitive).For the range finding test with chemical substances, the separation factor for the serial dilutions is usually ten (one order of magnitude difference between two successive dilutions).For effluents or waters, a 1:1 dilution factor is
49、normally applied (i.e. dilution of the previous concentration by half).Dilutions series for the definitive test on chemical substances are prepared with a separation factor not exceeding 3,2, whereas for effluents and waters, a 1:1 dilution factor is normally applied.The test is carried out with six replicates for each dilution plus a control (i.e. the test medium without sample) also in six replicates.When using a solvent in order to dissolve or disperse chemical substances, a preliminary test has to be
