1、BSI Standards PublicationBS ISO 19827:2016Water quality Determination of the acute toxicity to the freshwater rotifer Brachionus calyciflorusBS ISO 19827:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 19827:2016.The UK participation in its preparation was
2、 entrusted 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 B
3、ritish Standards Institution 2016. Published by BSI StandardsLimited 2016ISBN 978 0 580 87188 7ICS 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 29 Febr
4、uary 2016.Amendments issued since publicationDate Text affectedBS ISO 19827:2016 ISO 2016Water quality Determination of the acute toxicity to the freshwater rotifer Brachionus calyciflorusQualit de leau Dtermination de la toxicit aigue envers le rotifre deau douce Brachionus calyciflorusINTERNATIONA
5、L STANDARDISO19827First edition2016-02-15Reference numberISO 19827:2016(E)BS ISO 19827:2016ISO 19827:2016(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
6、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 or ISOs member body in the country of the requester.ISO copy
7、right officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 19827:2016ISO 19827:2016(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Test environment . 26
8、Reagents, test organisms and media . 27 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 test concentrations . 49.2 Preparation of the test and control solutions . 49.3
9、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 . 7Annex A (informative) Procedure for hatching of Brachionus calyciflorus cysts 9Annex B (informative) Preparation of artific
10、ial water 10Annex C (informative) Performance data .11Bibliography .13 ISO 2016 All rights reserved iiiContents PageBS ISO 19827:2016ISO 19827:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work
11、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-government
12、al, 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 document and those intended for its further maintenance are described in the I
13、SO/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 Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibil
14、ity 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 development of the document will be in the Introduction and/or on the ISO list of pa
15、tent 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 specific terms and expressions related to conformity assessment, as well as info
16、rmation 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, Subcommittee SC 5, Biological methods.iv ISO 2016 All rights reservedBS ISO
17、 19827:2016ISO 19827:2016(E)IntroductionThe evaluation of harmful effects on chemicals and pollutants on the biota in freshwater environments has for several years involved the performance of biological tests.Rotifers, and especially the species Brachionus calyciflorus, are of interest from the ecot
18、oxicological standpoint, 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 of the lethal effects of toxicants to the freshwater rotifer Brachionus calycif
19、lorus, after 24 h exposure. ISO 2016 All rights reserved vBS ISO 19827:2016BS ISO 19827:2016Water quality Determination of the acute toxicity to the freshwater rotifer Brachionus calyciflorusWARNING Persons using this document should be familiar with normal laboratory practice. This document does no
20、t 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.IMPORTANT It is absolutely essential that tests conducted in ac
21、cordance with this document be carried out by suitably qualified staff.1 ScopeThis International Standard specifies a method for the determination of the lethal effects of toxicants to Brachionus calyciflorus after 24 h exposure.The method is applicable to:a) chemical substances which are soluble, o
22、r which can be maintained as a stable suspension of dispersions under the conditions of the test;b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration or centrifugation;c) freshwaters;d) aqueous extracts.2 Normative referencesThe following documents, i
23、n whole or in part, are 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 Samplin
24、g Part 16: Guidance on biotesting of samplesISO 5814, Water quality Determination of dissolved oxygen Electrochemical probe methodISO/TS 20281, Water quality Guidance on statistical interpretation of ecotoxicity data3 Terms and definitionsFor the purposes of this document, the following terms and de
25、finitions apply.3.1control batchseries of replicates containing control solutionSOURCE: ISO 6341:2012, definition 3.13.2LC50concentration of dilution of the test sample which gives rise to 50 % mortality of the test organismsINTERNATIONAL STANDARD ISO 19827:2016(E) ISO 2016 All rights reserved 1BS I
26、SO 19827:2016ISO 19827:2016(E)3.3test batchseries of replicates filled with the same test solutionSOURCE: ISO 6341:2012, definition 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
27、 analysis and the percentage mortality of the test organisms is determined after 24 h exposure, with subsequent calculation of the 24 h LC50.The test is carried out in one or two stages: a “range-finding test” to determine the range of concentrations or dilutions needed for calculation of the 24 h L
28、C50; a “definitive test” conducted when the data of the range-finding test are not sufficient or adequate for calculation of the 24 h LC50.5 Test environmentThe test shall be carried out in the dark, in a temperature-controlled room or incubator at (25 1) C in the test containers.Maintain the atmosp
29、here 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 organisms and mediaUse only reagents of recognized analytical grade, unless otherwise specified.6.1 Test organismsThe t
30、ests organisms are females of the species Brachionus calyciflorus, obtained from a laboratory culture (see References 2,3,4), or hatched from commercially available cysts1).The procedure for hatching of Brachionus calyciflorus from cysts is described in Annex A.6.2 Culturing and dilution mediumA nat
31、ural or an artificial freshwater may be used as the water for stock culturing the rotifers, or as dilution water for the testing. Natural freshwater shall be collected from an unpolluted location; it must be filtered (30 m) and conditioned to test temperature and oxygen saturation prior to use. Natu
32、ral freshwater can be stored cold (4 1) C for several weeks.An example of artificial freshwater suitable for culturing and testing is given in Annex B.1) MicroBioTests Inc. Mariakerke, Belgium, is an example of a supplier able to provide suitable Brachionus calyciflorus cysts commercially. This info
33、rmation 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 All rights reservedBS ISO 19827:2016ISO 19827:2016(E)6.3 Reference substancePotassium dichromate (K2Cr2O7) or copper sulfate (CuSO45H2O) are recommended as ref
34、erence chemicals.NOTE Since K2Cr2O7is a carcinogenic substance, toxic via inhalation, the use of a ready-made solution with a defined concentration 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
35、 ApparatusUsual laboratory equipment and in particular the following.7.1 Temperature-controlled room or chamber7.2 Petri dishesSmall Petri dishes (diameter 5 cm) in glass or in inert plastic material.7.3 Test containersDisposable 48 wells (6 x 8) microplates made from chemically inert material.7.4 P
36、ipette for sampling rotifers, with a sufficient diameter for capturing the animals while allowing sampling of only a small volume of medium.For example single use 1 ml capillary mini-pipettes are suitable.7.5 Stereomicroscope with incident (bottom) illumination, with a magnification of at least 8 ti
37、mes and, if possible, a continuous magnification.7.6 Light source, providing a range of light intensity in the hatching Petri dish of 3 000 lx to 4 000 lx corresponding to 40 to 55 mol.m-2.s-1.7.7 Sample collecting bottles, as specified in ISO 5667-16.8 Treatment and preparation of samples8.1 Specia
38、l precautionsSpecial precautions are required for sampling, transportation, storage and treatment of freshwater or effluent.Sampling, 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 coll
39、ection. 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 to perform the test within 72 h the sample may be frozen and maintained deep-frozen (below 18 C) for testing within 2 months of collection, provided that characteristics
40、are known to be unaffected by freezing. At the time of testing, homogenize the sample to be analysed by shaking manually, and, 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
41、 the centre of the section of the test tube and half way between the surface of the deposited matters and the surface of the liquid.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
42、 document and does not constitute an endorsement by ISO of this product. ISO 2016 All rights reserved 3BS ISO 19827:2016ISO 19827:2016(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
43、.) filter or centrifuge the raw or decanted sample.The sample obtained by either of these methods is the sample submitted to testing.Measure the dissolved oxygen concentration (as specified in ISO 5814) and record the value in the test report.8.2 Preparation of the stock solutions of substances to b
44、e testedPrepare the stock solution of the substance to be tested by dissolving a known quantity of substance in a specified 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 und
45、er these conditions.For substances sparingly soluble in the test medium, refer to the specifications given in ISO 5667-16.9 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 geometri
46、c series with a separation factor which depends on the nature of the sample to be analysed (chemical substances, effluents, 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 10
47、 (one order of magnitude difference between two successive dilutions).For effluents or waters a 1:1 dilution factor is 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 exce
48、eding 3,2, whereas for effluents and waters a 1:1 dilution factor is normally applied.The test is carried out with 6 replicates for each dilution + a control (i.e. the test medium without sample) also in 6 replicates.When using a solvent in order to dissolve or disperse chemical substances, a prelim
49、inary test has to be performed to determine whether the highest concentration of the solvent used in the dilution series does not have a negative impact on the test organisms.9.2 Preparation of the test and control solutionsPrepare the test solutions by mixing the appropriate volumes of the sample to be tested (see Clause 8 and 9.1) or of its initial dilution, with dilution medium (6.2).Control and test solutions can be prepared in 10 ml containers (e.g. tubes in glass or in inert plastic material).The contai
