1、BSI Standards PublicationBS ISO 16778:2015W a t e r q u a l i t y C a l a n o i d copepod early-life stage test with Acartia tonsaBS ISO 16778:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 16778:2015. The UK participation in its preparation was entrusted
2、 to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British
3、Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 74917 9 ICS 13.060.70 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 20
4、15.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e d ISO 2015Water quality Calanoid copepod early-life stage test with Acartia tonsaQualit de leau Essai aux premiers stades de la vie de coppodes calanodes avec Acartia tonsiaINTERNATIONAL STANDARDISO 16778First edition 2015-0
5、6-15Reference number ISO 16778:2015(E)BS ISO 16778:2015ISO 16778:2015(E)ii ISO 2015 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2015, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by
6、 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 copyright officeCh. de Blandonnet 8 CP 4
7、01CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 16778:2015ISO 16778:2015(E)Foreword v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Apparatus . 26 Reagents 36.1 Water . 36.2 Culture and test media . 36.3 D
8、ilution water 37 Test organism . 38 Procedure. 48.1 Preparation of culture medium 48.2 Choice of test concentrations . 48.3 Preparation of test substance stock solution 48.4 Preparation of test solutions . 58.5 Incubation/Exposure 58.5.1 Test organisms and loading 58.5.2 Control of hatching . 58.5.3
9、 Larval development ratio (Early-life stages development). 68.5.4 Duration. 68.5.5 Handling of test vessels 68.5.6 Feeding . 68.5.7 Light and temperature 68.5.8 Aeration . 68.5.9 Dilution water and test solutions renewal or addition . 68.6 Measurements/Observations . 78.6.1 Concentration of the test
10、 substance 78.6.2 Physical-chemical parameters oxygen, pH, salinity, and temperature 89 Validity criteria 810 Expression of results 811 Denotion of results . 912 Interpretation of results . 913 Reference substance . 914 Test report . 9Annex A (informative) Defined culture and test media .11Annex B (
11、informative) Specific details on renewal of test solution, feeding regime, and an example of a flow diagram for a larval development test (early-life stage test) with Acartia tonsa 13Annex C (informative) Sampling for chemical analyses 17Annex D (informative) Lugols solution 18Annex E (informative)
12、Data collection sheet for Acartia tonsa early-life stage test .19Annex F (informative) Calculations 21Annex G (informative) Culturing of Acartia tonsa 23 ISO 2015 All rights reserved iiiContents PageBS ISO 16778:2015ISO 16778:2015(E)Annex H (informative) Specific details on secondary sexual characte
13、ristics for Acartia tonsa .28Annex I (informative) General safety precautions and techniques for handling environmental samples 30Bibliography .36iv ISO 2015 All rights reservedBS ISO 16778:2015ISO 16778:2015(E)ForewordISO (the International Organization for Standardization) is a worldwide federatio
14、n 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.
15、 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 document and thos
16、e 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 Directives, Part 2 (s
17、ee 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 development of the doc
18、ument 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 specific terms an
19、d 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, Subcommittee
20、 SC 5, Biological methods. ISO 2015 All rights reserved vBS ISO 16778:2015ISO 16778:2015(E)BS ISO 16778:2015Water quality Calanoid copepod early-life stage test with Acartia tonsaWARNING Persons using this document should be familiar with normal laboratory practice. This International Standard does
21、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.IMPORTANT It is absolutely essential that tests conducted in
22、accordance with this document be carried out by suitably qualified staff.1 ScopeThis International Standard specifies an early-life stage test procedure for determination of the toxic effects of a chemical substance, effluent, or water sample on a cold-water marine and brackish water copepod species
23、 under semi-static conditions. The biological endpoints include survival and development of the early-life stages. The exposure starts with eggs and is continued until emergence of juvenile stages.Copepods occur widely in marine, brackish, and fresh water ecosystems. They represent important prey it
24、ems for the larvae of many fish and larger invertebrates and are increasingly used as a live food source in aquaculture. They feed on phytoplankton and, thus, are an ecologically important energy-transfer link between primary producers and higher trophic levels.2 Normative referencesThere are no nor
25、mative references cited in this document.3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1ECeffect concentration3.2ECxcalculated concentration from which an effect of x % is expected3.3larval development ratioLDRfraction of animals that have turn
26、ed into a copepodite stage compared to the total number of surviving nauplii and copepodites within a given period of time (5 d to 6 d)3.4lowest observed effect concentrationLOEClowest concentration within the experimental range at which a significant effect is observedINTERNATIONAL STANDARD ISO 167
27、78:2015(E) ISO 2015 All rights reserved 1BS ISO 16778:2015ISO 16778:2015(E)3.5no observed effect concentrationNOECtested concentration just below the LOECSOURCE: ISO/TS 20281:2006, 3.183.6x % confidence intervalsinterval of values within which the measured or calculated value is likely to be present
28、 with a probability of x %3.7dilution waterwater with defined properties (e.g. salinity) or natural seawater used for stepwise dilution of the test sample or used as control4 PrincipleThe test is an early-life stage test, where the organisms are exposed to various concentrations of a chemical substa
29、nce, effluent, or water sample, from the egg stage to the juvenile stages. Survival and development of early-life stages larval development ratio (LDR) are dependent on the investigated parameters. The total test duration is about 5 d to 6 d, which is sufficient time to investigate the development f
30、rom nauplii to copepodites.The naupliar (larval) and copepodite (juvenile) stages are morphologically distinct, and therefore, the transition from the last naupliar to the first copepodite stage is easily observed. Larval development ratio (LDR) is recorded after 5 d to 6 d, when about 60 % of the c
31、ontrol animals have reached a copepodite stage, and is expressed as the ratio of copepodites to the total number of surviving early-life stages (nauplii + copepodites) at the end of the LDR test. Hatching success and mortality of the early stages should be presented along with the LDR.The outcome of
32、 the test is either the no observed effect concentration and the lowest observed effect concentration (NOEC-LOEC) values or the effect concentrations with a certain degree (x %) of inhibition (ECx) (e.g. EC50and EC10).5 ApparatusTest vessels and other apparatus, which will come into contact with the
33、 dilution water and test solutions, should be made entirely of glass or other materials chemically inert to the test chemical.5.1 Standard laboratory apparatus, e.g. measurements of pH, dissolved oxygen concentration, salinity, and temperature.5.2 Glass flasks, volume 1 l, 2 l, and 5 l.5.3 Air pumps
34、.5.4 Air filters, pore size 0,2 m.5.5 Peristaltic pump for food supply.5.6 Temperature-controlled cabinet or room.5.7 Low-magnifying stereomicroscope, preferably with dark field illumination.2 ISO 2015 All rights reservedBS ISO 16778:2015ISO 16778:2015(E)5.8 Apparatus for membrane filtration.5.9 Fil
35、ters, 0,2 m (6.2) and eventually filters with grids (8.5.1).5.10 Nets, mesh sizes 50 m and 180 m to 200 m, for isolation of eggs, for capture and transfer of animals, and as filters when medium is changed.5.11 Adequate apparatus for the control of the lighting regime.6 Reagents6.1 WaterAll water use
36、d in preparation of culture medium shall be clean natural seawater or deionized water or of equivalent purity. 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.6.2 Culture and test media
37、Culture and test media are prepared from either reconstituted salt water or filtered (0,2 m) natural marine water from an unpolluted location. An example of reconstituted salt water suitable for cultivation and testing is given in Annex A. Reconstituted salt water media with a known composition in w
38、hich the copepods show suitable long-term survival, normal behaviour, development, and fecundity can be used as culture and test media, i.e. dilution water.6.3 Dilution waterThe salinity of the dilution water should be the same as that of the culture medium (see Annex A). The dilution water shall ha
39、ve a dissolved oxygen concentration above 70 % of the air saturation value and a pH of 8,0 0,3 before being used to prepare the test solutions. If there is evidence of marked change of pH at the highest test concentration, it is advisable to adjust the pH of the stock solution/environmental sample t
40、o that of the dilution water before preparing the dilution series. The pH adjustment of the stock solution or test concentrations shall not change the concentration to any significant extent or lead to chemical reaction or precipitation of the test substance. HCl and NaOH are preferred for pH adjust
41、ments.If the physical conditions or the salinity of the salt water to be used in the test differ more than 5 C in temperature or 10 salinity from those used for routine culturing, it is good practice to include an adequate pre-test acclimation period at the same temperature (20 1) C and salinity (20
42、 2) of 2 to 3 weeks to avoid stressing the eggs and animals. Use of another temperature or salinity, which can be more appropriate in oceanic or brackish water situations, shall be justified in the test report.7 Test organismThe species to be used is the marine calanoid copepod Acartia tonsa Dana (s
43、ee Annex G and Annex H).Eggs used in the test should be collected from a healthy stock (i.e. showing no signs of stress such as high mortality, poor fecundity, etc.). The stock animals shall be maintained in culture conditions (light, temperature, medium, and feeding) similar to those to be used in
44、the test (culturing method for A. tonsa is described in Annex G). ISO 2015 All rights reserved 3BS ISO 16778:2015ISO 16778:2015(E)8 Procedure8.1 Preparation of culture mediumNatural seawater or a reconstituted medium can be used. A suitable reconstituted culture medium is described in Annex A. Howev
45、er, alternative reconstituted media can be used as long as the validity criteria for the test are met (see Clause 9). The defined medium described in Annex A contains a chelating agent and therefore, might not be appropriate for testing of samples that contain metals. The salinity can be varied by c
46、hoosing a desired amount of the 10 % salinity solution. The salinity of natural seawater and environmental samples can be raised by using the same 10 % salinity solution (Table A.1) or lowered by adding an appropriate volume of M7 (see Reference 1 and Annex A) or deionized water.8.2 Choice of test c
47、oncentrationsPrior knowledge of the toxicity of the test substance (e.g. from an acute test1) or from range-finding studies) should help in selecting appropriate test concentrations. As a rule of thumb, the highest concentration in the early-life stage test should be chosen within the interval betwe
48、en LC10and LC20of the acute 48 h test to avoid significant effect on survival.At least 5 different concentrations should be tested in a geometric series with a factor between concentrations not exceeding 3,2. Justification should be provided if fewer than five concentrations are used. Substances sho
49、uld not be tested above their solubility limits in dilution water. A dilution water control shall be included and, if a solvent is used, a solvent-control shall also be included (8.3), containing the same concentration of solvent as the test series.If there are substantial reason to assume that a high concentration of a chemical or an environmental sample will have low/no toxicity at a high concentration (e.g. at 10 mg/l or 100 ml/l), the early-life stage test can be performed as a limit test, using a test concentration
