1、PD ISO/TS 18220:2016 Water quality Larval development test with the harpacticoid copepod Nitocra spinipes BSI Standards Publication WB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06PD ISO/TS 18220:2016 PUBLISHED DOCUMENT National foreword This Published Document is the UK implementation of IS
2、O/TS 18220:2016. The UK participation in its preparation was entrusted to Technical Committee 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
3、contract. Users are responsible for its correct application. The British Standards Institution 2016. Published by BSI Standards Limited 2016 ISBN 978 0 580 82028 1 ICS 13.060.70 Compliance with a British Standard cannot confer immunity from legal obligations. This Published Document was published un
4、der the authority of the Standards Policy and Strategy Committee on 30 June 2016. Amendments/corrigenda issued since publication Date T e x t a f f e c t e dPD ISO/TS 18220:2016 ISO 2016 Water quality Larval development test with the harpacticoid copepod Nitocra spinipes Qualit de leau Essai de dvel
5、oppement larvaire avec le coppode harpacticode Nitocra spinipes TECHNICAL SPECIFICATION ISO/TS 18220 Reference number ISO/TS 18220:2016(E) First edition 2016-07-01PD ISO/TS 18220:2016ISO/TS 18220:2016(E)ii ISO 2016 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in Switzerland A
6、ll rights reserved. Unless otherwise specified, 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
7、from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Ch. de Blandonnet 8 CP 401 CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Fax +41 22 749 09 47 copyrightiso.org www.iso.orgPD ISO/TS 18220:2016ISO/TS 18220:2016(E)Foreword iv In
8、troduction v 1 Scope . 1 2 T erms and definitions . 1 3 Principle 2 4 Reagents 2 5 Cultivation . 3 5.1 Test organism . 3 5.2 Algae for feeding 3 6 Apparatus . 3 7 Procedure. 4 7.1 Production of nauplii to be used in test . 4 7.2 Choice of test concentrations . 4 7.2.1 Hypothesis testing 5 7.2.2 Regr
9、ession analysis 5 7.3 Preparation of solutions to be used in test . 5 7.3.1 Stock solution . 5 7.3.2 Test solutions 5 7.4 Start of test . 6 7.5 Incubation/exposure . 6 7.6 Maintenance 6 7.7 Measurements/observations . 6 7.7.1 Mortality . 6 7.7.2 Larval development ratio (LDR). 7 7.7.3 Physical-chemi
10、cal parameters oxygen, pH and salinity 7 7.7.4 Concentration of the test substance 7 8 Validity criteria 7 9 Evaluation of results . 8 9.1 Calculation of results . 8 9.2 Expression of results 8 9.3 Interpretation of results . 8 10 Reproducibility 8 11 Test report . 8 Annex A (informative) Biology an
11、d cultivation of Nitocra spinipes .10 Annex B (informative) Nitocra spinipes larval development ratio .15 Annex C (informative) Calculations 17 Bibliography .19 ISO 2016 All rights reserved iii Contents PagePD ISO/TS 18220:2016ISO/TS 18220:2016(E) Foreword ISO (the International Organization for Sta
12、ndardization) is a worldwide 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 righ
13、t to be represented on that 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
14、 used to develop this document 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 rul
15、es of the ISO/IEC Directives, 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 ident
16、ified during the development 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
17、 on the meaning of ISO specific terms and expressions related to conformit y assessment, as well as information about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html. The committee responsib
18、le for this document is ISO/TC 147 , Water quality, Subcommittee SC 5, Biological methods.iv ISO 2016 All rights reservedPD ISO/TS 18220:2016ISO/TS 18220:2016(E) Introduction Harpacticoid copepods are predominantly benthic, occurring widely in marine, brackish and fresh water ecosystems. They repres
19、ent important prey items for the benthic larvae of many fish species and larger invertebrates and constitute an ecologically important energy-transfer link between the organic phase of the sediment and higher trophic levels. The euryhaline brackish water harpactoid Nitocra spinipes (Crustacea) is a
20、common component of the benthic meiofana in shallow coastal waters around the world (see Reference 6). ISO 2016 All rights reserved vPD ISO/TS 18220:2016PD ISO/TS 18220:2016Water quality Larval development test with the harpacticoid copepod Nitocra spinipes W A R N I N G P e r s o n s u s i n g t h
21、i s T e c h n i c a l S p e c i f i c a t i o n sh o u l d b e f a m i l i a r w i t h n o r m a l l a b o r a t o r y p r a c t i c e . T h i s T e c h n i c a l S p e c i f i c a t i o n d o e s n o t p u r p o r t t o a d d r e s s a l l o f t h e safety problems, if any, associated with its use.
22、 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 Technical S p e c i f i c a t i o n b e c a r r i e d o u t b
23、 y s u i t a b l y q u a l i f i e d s t a f f . 1 Scope This Technical Specification specifies an early-life stage procedure for determination of the toxic effects of chemicals and water samples on a cold-water brackish water copepod species under semi- static conditions. The biological test variab
24、les include survival and development of the early-life stages. The exposure starts with newly hatched (24 h) nauplii (larvae) and is continued until emergence of (c. 50 %) copepodites (juveniles) in the control. The benthic living Nitocra complements the planktonic Acartia species in ISO 16778. Thes
25、e organisms represent different life-history strategies as Nitocra is egg-carrying, whereas Acartia is a broadcasting calanoid copepod and thus, different sensitivities of specific life stages. Nitocra is a fresh to brackish water species, which allows testing low salinity waters and is complementar
26、y to A. tonsa, which is of marine origin and poorly tolerates low salinities. 2 Terms a nd definiti ons For the purposes of this document, the following terms and definitions apply. 2.1 nauplii larvae 2.2 copepodites juveniles 2.3 larval development ratio LDR ratio of copepodites (2.2) to the total
27、number of surviving early-life stages (nauplii + copepodites) at the end of the test 2.4 lowest observed effect concentration LOEC lowest concentration within the experimental range at which a significant effect is observed 2.5 no observed effect concentration NOEC tested concentration just below th
28、e LOEC (2.4) TECHNICAL SPECIFICATION ISO/TS 18220:2016(E) ISO 2016 All rights reserved 1PD ISO/TS 18220:2016ISO/TS 18220:2016(E) 2.6 effect concentration EC x calculated concentration from which an effect of x % is expected 2.7 mortality calculated on dead and missing animals at the end of the test
29、divided by animals at start 2.8 c on f ide nc e i nt e r v a l A x % interval of values within which the measured or calculated value is likely to be present with a probability of x % 2.9 salinity S dimensionless value of which, for the purpose of checking water quality, may be regarded as an estima
30、te of the concentration, in grams per kilogram, of dissolved salts in sea water Note 1 to entry: It is defined algorithmically, in terms of the ratio (K15) of the electrical conductivity of the sample, at 15 C and 1 atm, to that of defined potassium chloride solution (32,436 6 g/kg of sample) at the
31、 same temperature and pressure. 3 Principle The test is an early-life stage test, in which the organisms are exposed to various concentrations of a test substance or water sample under semi-static test conditions from the first naupliar stage (N-1) to the first copepodite stages (C-1, C-2, etc.). Su
32、rvival and development of early-life stages larval development ratio (LDR), are the investigated test variables. The exposure starts with newly hatched (24 h) nauplii (larvae) and is continued until the emergence of (approximately 50 %) copepodites (juveniles) in the control. The total test duration
33、 is about 6 d to 7 d, which is sufficient time to investigate the development from N-1 to 50 % copepodites in the control. 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
34、 observed. The outcome of the test is either the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values or the effect concentrations with a certain degree (x %) of inhibition (EC x ) (e.g. EC 50and EC 10 ). 4 Reagents 4.1 Test organism The species to be us
35、ed is the brackish water harpacticoid copepod Nitocra spinipes Boeck. Newly hatched (less than 24 h of age) nauplii 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 (l
36、ight, temperature, medium and feeding) similar to those to be used in the test (culturing method for N. spinipes is described in Annex A).2 ISO 2016 All rights reservedPD ISO/TS 18220:2016ISO/TS 18220:2016(E) 4.2 Water, deionized or of equivalent purity, to prepare artificial sea water or to dilute
37、natural sea water. 4.2.1 Artificial sea w at er An example of artificial sea water suitable for cultivation and testing is included in Annex A. Any artificial sea water with a known composition in which the copepods show suitable long-term survival, normal behaviour, development and fecundity may be
38、 used as culture and dilution medium (4.3). 4.2.2 Natural sea water Natural sea water shall be collected from an unpolluted location. Any natural sea water with a known composition in which the copepods show suitable long-term survival, normal behaviour, development and fecundity may be used as cult
39、ure and dilution medium. Suspended particles shall be 10 mg/l and can be stored cold for approximately 6 months before preparation of culture or dilution medium. 4.3 Medium 4.3.1 Culture medium Culture medium is used for cultivating Nitocra spinipes and is prepared from either natural or artificial
40、sea water (4.2). Natural sea water shall be filtered (30 m) and heated to 80 C to kill undesired organisms and then conditioned (24 h) to culture temperature and oxygen saturation. The culture medium can be stored cold for several weeks. 4.3.2 Dilution medium Dilution medium is used for diluting wat
41、er samples or dissolving chemicals and is prepared from culture medium that has first been filtered (GF/C-1,2 m) before use. Salinity of the dilution medium should be the same as the culture medium. Salinities between 3 and 25 can be used. The dilution medium shall have a dissolved oxygen concentrat
42、ion above 70 % of the air saturation value and a pH of 7,5 1,0 before being used to prepare the test solutions. If the physical conditions or the salinity of the medium to be used in the test differ more than 5 C or 10 % from those used for routine culturing, it is good practice to include an adequa
43、te cultivating acclimation period at the same salinity (2 ) of 2 weeks to 3 weeks to avoid stressing of the larvae. 5 Cultivation 5.1 Test organism See Annex A for detailed information. 5.2 Algae for feeding See Annex A for detailed information. 6 Apparatus All equipment, which will come in contact
44、with the test medium, shall be made of glass or chemically inert material. 6.1 Glass vessels, approximately 150 ml, diameter 8 cm, and height 4,5 cm, for Nitocra spinipes cultivation. ISO 2016 All rights reserved 3PD ISO/TS 18220:2016ISO/TS 18220:2016(E) 6.2 Test vessels, approximately 15 ml, diamet
45、er 2,5 cm, height 4 cm, with flat bottom. 6.3 pH meter. 6.4 Oxygen meter. 6.5 Conductivity meter. 6.6 Wide pipettes, for sampling animals, preferably salinized to prevent copepods from adhering to pipette walls. 6.7 Temperature-control cabinet or room, (22 1) C. 6.8 Low-magnifying stereo microscope.
46、 6.9 Inverted microscope. 6.10 A ppar atus for memb r ane filtr ation. 6.11 Filters, 1,2 m and 30 m. 7 Procedure 7.1 Production of nauplii to be used in test Nauplii aged less than 24 h are used for initiating a test. To produce a sufficient number of nauplii, the procedure presented below should be
47、 followed. The day before the test starts, approximately 300 females with well-developed egg sacs are sampled by pipette under a low-magnifying stereomicroscope and randomly transferred (approximately 60 in each) to six glass “hatch” vessels containing 100 ml dilution medium. The isolated female cop
48、epods are fed with a suspension of Rhodomonas salina to a concentration of 2,5 10 5cells/ml. 7.2 Choice of test concentrations The range of the test concentrations should preferably not include any concentrations that have a significant effect on survival since the main objective of the test is to m
49、easure sublethal effects (i.e. development). Prior knowledge of the toxicity of the test substance, i.e. from an acute test (see Reference 2) 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 set at 10 % to 20 % of the acute 96 h-LC 50to avoid significant effect on survival. At least five different concentrations should be tested in a geometric series with a factor between concentrations not
