1、BSI Standards PublicationBS ISO 17244:2015W a t e r q u a l i t y D e t e r m i n a t i o nof the toxicity of watersamples on the embryo-larvaldevelopment of Japaneseoyster (Crassostrea gigas)and mussel (Mytilus edulis orMytilus galloprovincialis)BS ISO 17244:2015 BRITISH STANDARDNational forewordTh
2、is British Standard is the UK implementation of ISO 17244:2015.The UK participation in its preparation was 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
3、include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 75490 6ICS 13.060.70Compliance with a British Standard cannot confer immunity fromlegal obligations.This B
4、ritish Standard was published under the authority of theStandards Policy and Strategy Committee on 30 September 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 17244:2015 ISO 2015Water quality Determination of the toxicity of water samples on the embryo-larval d
5、evelopment of Japanese oyster (Crassostrea gigas) and mussel (Mytilus edulis or Mytilus galloprovincialis)Qualit de leau Dtermination de la toxicit dchantillons aqueux sur le dveloppement embryo-larvaire de lhutre creuse (Crassostrea gigas) et de la moule (Mytilus edulis ou Mytilus galloprovincialis
6、)INTERNATIONAL STANDARDISO17244First edition2015-09-15Reference numberISO 17244:2015(E)BS ISO 17244:2015ISO 17244:2015(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 r
7、eproduced 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 or ISOs member body in the country of the reque
8、ster.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 17244:2015ISO 17244:2015(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Test orga
9、nisms and seawater 25.1 Spawning stock or mature bivalves. 25.2 Reference seawater . 35.2.1 Natural seawater 35.2.2 Synthetic seawater . 35.2.3 Hypersaline brine (HSB) . 46 Equipment . 47 Reference substance . 58 Test procedure 58.1 Collection, preparation, and preservation of aqueous samples . 58.2
10、 Preparation of test samples . 58.2.1 Chemicals . 58.2.2 Aqueous samples . 58.3 Selection of concentration/dilution range 68.4 Collecting gametes . 68.4.1 General 68.4.2 Thermal stimulation . 68.4.3 Stripping the gametes 88.5 Measurement of egg density . 88.6 Fertilization and inoculation of fertili
11、zed eggs . 88.7 Incubation . 98.8 Observation 98.9 Analytical measurements . 109 Expression of results .1010 Validity criteria .1211 Test report 12Annex A (informative) Overview of the test applied to the Japanese oyster Crassostrea gigas .13Annex B (informative) Performance data 14Bibliography .22
12、ISO 2015 All rights reserved iiiContents PageBS ISO 17244:2015ISO 17244:2015(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through IS
13、O 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-governmental, in liaison with ISO, also take part in the work. ISO collaborates c
14、losely 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 ISO/IEC Directives, Part 1. In particular the different approval criteri
15、a 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 possibility that some of the elements of this document may be the subject of pa
16、tent 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 patent declarations received (see www.iso.org/patents).Any trade name use
17、d in this document is information given for the convenience of users and does not constitute an endorsementFor an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barr
18、iers to Trade (TBT), see the following URL: Foreword Supplementary Information .The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.iv ISO 2015 All rights reservedBS ISO 17244:2015ISO 17244:2015(E)IntroductionTraditionally, the level of pol
19、lution affecting a marine environment is shown in terms of the concentration levels of the contaminants present in the environment of interest. However, these measurements do not provide an estimation of the harmful effects on organisms and have to be complemented with the biological responses obtai
20、ned through biotests (see Reference 5).Among the marine organisms used to assess the potential impact of chemicals or discharges into the environment, bivalve embryos and larvae are, together with sea urchins, among the organisms which are most frequently used in biotests. This has been the case sin
21、ce the first research undertaken by Lillies (1921) (see Reference18) on the sea urchin Arbacia and by Prytherch (1924) (see Reference21) on the oyster Crassostrea virginica. The embryos and larvae are less tolerant to pollutants than the adults of the same species. They therefore represent the criti
22、cal stages for the toxicity tests (see References19 and 30). Since 1972, Woelke (see Reference35) has recommended the use of the Pacific oyster, Crassostrea gigas, to assess the quality of seawater. Furthermore, their worldwide distribution in coastal waters, as well as their commercial importance (
23、see Reference10), make bivalves the species of choice for the undertaking of biotests.The results of these biotests demonstrate the necessity to determine the potential toxicity thresholds of chemicals which could enter the marine environment either accidentally or chronically, as well as the “biolo
24、gical quality” of an environment or the potential toxicity of river water or a discharge that reach the sea. Quiniou et al. (1993, 1997) (see References27 and 26) and His et. al (1999) (see Reference 11) defined potential toxicity on the basis of teratological effects.This International Standard spe
25、cifies a method based on the embryo-larval development of bivalves (oyster or mussel). It can be routinely used to assess development abnormalities caused by the possible presence of chemicals and mixtures in seawater. It also allows to assess the toxicity of aqueous samples like seawater, surface w
26、ater, effluents (urban, agricultural, industrial effluents, etc.), aqueous extracts from sediments, and petroleum products that could be leached in the water column at the time of their resuspension or discharge and presence in the sea.This test can be performed throughout the year with mature bival
27、ves sampled from the natural environment during their reproduction periods or mature bivalves which come from a hatchery where they have been conditioned.This toxicity test, recommended by the International Council for the Exploration of the Sea (ICES), (see Reference14), has been the subject of the
28、 first European inter-calibration test performed in 1991 (see Reference 31). The protocol described in this International Standard corresponds to a modification and simplification of the ASTM standard method (1994) (see Reference3).The toxicity assessment of metals performed on C. gigas and Mytilus
29、edulis demonstrated that both organisms had a similar level of sensitivity (see References19 and 15). Two other studies performed on urban effluents showed similar findings for both species (see References16 and 28). These observations have been confirmed by the work carried out on mercury by Beiras
30、 and His (1994) (see Reference4), who compared the findings of four embryo-larval tests: M. edulis, M. galloprovincialis, C. gigas, and C. virginica. Another study showed that the embryos of C. gigas are more sensitive to metals and hydrocarbons than the other marine organisms which are commonly use
31、d, for example, polychaete, amphipods, fish, and crustaceans (see Reference 8).The sensitivity of the bivalve embryo-larval development confirms the suitability of this test to assess the toxicity of chemicals and aqueous samples. The pH, salinity, and temperature ranges acceptable to bivalves make
32、them easy to use in ecotoxicity studies, particularly when assessing the quality of coastal and estuarine environments (see Reference11). ISO 2015 All rights reserved vBS ISO 17244:2015BS ISO 17244:2015Water quality Determination of the toxicity of water samples on the embryo-larval development of J
33、apanese oyster (Crassostrea gigas) and mussel (Mytilus edulis or Mytilus galloprovincialis)WARNING Persons using this International Standard should be familiar with normal laboratory practice. This International Standard does not purport to address all of the safety problems, if any, associated with
34、 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 International Standard be carried out by suitably qua
35、lified staff.1 ScopeThis International Standard specifies a method for determining the effects of chemical and aqueous samples on the embryo-larval development of marine bivalves. It allows the determination of the concentration levels that result in an abnormality in embryo-larval development. This
36、 test is suitable for salinity ranges between 20 and 40 for mussels and between 25 and 35 for oysters. This method applies to chemical substances and preparations, marine and brackish waters, streams and aqueous effluents (urban, agricultural, industrial effluents, etc.) as long as the salinity is a
37、djusted and/or dilution is limited so that the aforementioned salinity ranges are respected, and aqueous extracts (pore water, elutriates, eluates, and leachates) from sediments and petroleum products.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in t
38、his 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 biotesting of samplesISO 144
39、42, Water quality Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1ECxcalculated concentration (of a substance) or dilution (of
40、an aqueous sample, in %) for which an effect of x % is expected compared to the controlINTERNATIONAL STANDARD ISO 17244:2015(E) ISO 2015 All rights reserved 1BS ISO 17244:2015ISO 17244:2015(E)3.2lowest observed effect concentrationLOEClowest concentration of the tested sample at which a statisticall
41、y significant effect is observed3.3no observed effect concentrationNOECtested concentration just below the LOECSOURCE: ISO/TS 20281:2006, 3.183.4reference seawaternatural or synthetic seawater used to induce gamete production and prepare the solutions to be tested3.5D-shell stage larvaelarvae stage
42、so named due to their characteristic D-shape under microscopic examinationNote 1 to entry: The normal D larvae obtained after incubation have fully developed and symmetrical shells with a straight hinge. The larvae size is regular and around 70 m. After fixation, the mantle nearly fills the interior
43、 of the larvae, but is totally included within the two closed shells.4 PrincipleThis biotest assesses the effects of chemicals and aqueous environmental samples on the embryo-larval development of marine bivalves under static conditions.The exposure is performed from fertilized eggs to D larvae. Thi
44、s static test aims to determine the concentration level (ECx) which results in abnormalities for x % of exposed larvae in 24 h for the Japanese oyster, also named Pacific oyster (Crassostrea gigas), and in 48 h for the mussel (Mytilus edulis or Mytilus galloprovincialis). Several parameters can be a
45、ssessed on the abnormal larvae: alteration of the shell (hinge is not straight, unequal, or incomplete valves), hypertrophy of the mantle, delayed or stopped embryonic development, and finally, death. The results are expressed as ECx(EC20or EC50). The lowest observed effect concentration (LOEC) and
46、no observed effect concentration (NOEC) can also be determined.NOTE This method can be applied to other species of bivalves (e.g. C. virginica). Nevertheless, the test conditions have to be defined to reach the validity criteria of the standard.5 Test organisms and seawater5.1 Spawning stock or matu
47、re bivalvesThe mature bivalves used for gamete production can be obtained from the natural environment during reproductive periods as long as the good quality of their sampling area has been proven. The reproduction period along the European and African coasts depends on the site. In some places, it
48、 can occur all year.For oysters, it is also possible to use mature animals from hatcheries where they have previously undergone a conditioning cycle so that they are ready for spawning as soon as they arrive in the laboratory. This enables to perform tests throughout the year.As soon as the bivalves
49、 are received in the laboratory, it is recommended to keep them dry until the beginning time of the experiment (for example, 15 C) or in seawater at a temperature close to their conditioning or rearing temperature (for example, 20 C for hatchery oysters). If the mature bivalves have to be kept for more than 48 h after sampling and/or dispatching, they shall be stored in water (see 5.2) with the same temperature as the original location and shall be provided with rich and appropriate 2 ISO 2
