1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS ISO 14371:2012W a t e r q u a l i t y D e t e r m i n a t i o n of fresh water sediment toxicity to Heterocyprisincongruens (Crustacea, Ostracoda)BS ISO 14371:2012 BRITISH STA
2、NDARDNational forewordThis British Standard is the UK implementation of ISO 14371:2012.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 publicat
3、ion does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 69872 9ICS 13.060.70Compliance with a British Standard cannot confer immunity from
4、legal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 March 2012.Amendments issued since publicationDate Text affectedBS ISO 14371:2012 ISO 2012Water quality Determination of fresh water sediment toxicity to Heterocypris incongr
5、uens (Crustacea, Ostracoda)Qualit de leau Dtermination de la toxicit des sdiments deau douce envers Heterocypris incongruens (Crustacea, Ostracoda)INTERNATIONAL STANDARDISO14371First edition2012-03-01Reference numberISO 14371:2012(E)BS ISO 14371:2012ISO 14371:2012(E)ii ISO 2012 All rights reservedCO
6、PYRIGHT PROTECTED DOCUMENT ISO 2012All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address belo
7、w or ISOs member body in the country of the requester.ISO copyright officeCase postale 56 CH-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 749 09 47E-mail copyrightiso.orgWeb www.iso.orgPublished in SwitzerlandBS ISO 14371:2012ISO 14371:2012(E) ISO 2012 All rights reserved iiiContents PageForeword
8、 ivIntroduction v1 Scope 12 Normative references . 13 Terms and definitions . 14 Principle . 15 Test environment . 26 Reagents, test organisms and media 27 Apparatus and material . 38 Special precautions for sampling, transportation, storage and treatment of the sediment samples . 49 Procedure 49.1
9、Hatching of the cysts . 49.2 Pre-feeding of the ostracod neonates . 49.3 Length measurement of the neonates . 49.4 Addition of sediment and algal food to the test container . 59.5 Transfer of the ostracod neonates into the test container . 59.6 Incubation of the test system . 69.7 Measurements 610 R
10、eference test 811 Tests on pure chemicals or preparations .1012 Validity criteria .1013 Test report .10Annex A (informative) Culturing of Heterocypris incongruens for cyst production 11Annex B (informative) Precision data13Bibliography .15BS ISO 14371:2012ISO 14371:2012(E)ForewordISO (the Internatio
11、nal 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 ISO technical committees. Each member body interested in a subject for which a technical committee has been
12、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 closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standar
13、dization.International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for vot
14、ing. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.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
15、 patent rights.ISO 14371 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.iv ISO 2012 All rights reservedBS ISO 14371:2012ISO 14371:2012(E)IntroductionThe evaluation of harmful effects on water quality has for several years involved the performance
16、 of biological tests. Historically, toxicity tests have mainly focused on the impact of pollutants present in the water column of aquatic ecosystems, without considering the hazard of toxicants present and accumulating in the sediments.“Direct contact” tests in which the test organisms are exposed t
17、o the whole sediment have been gradually developed with endobenthic species, such as chironomid larvae Chironomus riparius or Chironomus dilutus (formerly C. tentans) or epibenthic amphipod crustaceans (Hyalella azteca).The test specified in this International Standard is a direct contact test for d
18、etermination of the percentage mortality and/or growth inhibition on the fresh water ostracod Heterocypris incongruens (Ramdohr, 1808) after 6 d exposure to a whole sediment (see References 1, 2).H. incongruens is a cosmopolitan ostracod species, which has to date already been used extensively for t
19、oxicity testing not only of whole sediments, but also by extension on sludges and soils (see References 321).The direct contact test with H. incongruens has a sensitivity which is quite similar to that of the amphipod crustacean Hyalella azteca and the midge larva Chironomus riparius (see References
20、 2225).The assays are performed with neonates hatched from dormant eggs (cysts), which bypasses the need for culturing or maintaining live stock cultures of test organisms.H. incongruens neonates (150 m to 200 m) are substantially smaller than Hyalella azteca and Chironomus riparius, and the assays
21、can be performed in much smaller test containers, hence require much less bench space and incubator space.The effects are evaluated after a shorter exposure time (6 d) than in the assays with the amphipod crustacean (10 d to 28 d) and midge larvae species (10 d to 28 d). ISO 2012 All rights reserved
22、 vBS ISO 14371:2012BS ISO 14371:2012Water quality Determination of fresh water sediment toxicity to Heterocypris incongruens (Crustacea, Ostracoda)WARNING Persons using this International Standard should be familiar with normal laboratory practice. This standard does not purport to address all of th
23、e 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 accordance with this Internation
24、al Standard be carried out by suitably qualified staff.1 ScopeThis International Standard specifies a method for the determination of lethal as well as sublethal effects of contaminated sediments on the ostracod crustacean Heterocypris incongruens after 6 d exposure.The method is applicable not only
25、 to fresh water sediments, but also by extension to solid wastes and soils after addition of (uncontaminated) water.The method can also be applied to chemicals or preparations which are spiked into a reference sediment.This International Standard is not applicable to the testing of sediments from th
26、e estuarine or marine environment.2 Normative referencesThe following documents, in 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 referen
27、ced document (including any amendments) applies.ISO 5667-15:2009, Water quality Sampling Part 15: Guidance on the preservation and handling of sludge and sediment samples ISO 5667-16, Water quality Sampling Part 16: Guidance on biotesting of samples 3 Terms and definitionsFor the purposes of this do
28、cument, the following terms and definitions apply.3.1neonatenewly hatched individual3.2pre-feedingfeeding of the neonates (3.1) with a small amount of dried algae (Spirulina) prior to the test4 PrincipleFreshly hatched Heterocypris incongruens larvae are exposed to the sediment sample under analysis
29、 and the percentage mortality of the test organisms is determined after 6 d exposure.If the percentage mortality is low (e.g. 120 nauplii). 10 mg dry ostracod cysts is normally sufficient.4 ISO 2012 All rights reservedBS ISO 14371:2012ISO 14371:2012(E)Table 1 Data report template 1: Length of ostrac
30、od neonatesOstracod neonateLengthm12345678910Mean length, Lstart9.4 Addition of sediment and algal food to the test containerPerform the direct contact test on the whole sediment in six replicates, in parallel to a control sediment (6.6) also in six replicates. When using six-well microplates (7.3),
31、 the assay requires two such plates.Inoculate three wells in each microplate with whole sediment and the three other wells with control sediment. Alternatively, the six wells of the first microplate can be filled with whole sediment, and those of the second plate with control sediment.Put 2 ml test
32、medium (6.3) into all the cups of the two microplates.Fill the cup of the spatula scoop (7.6) with test sediment and strike off the excess of sediment with the flat spatula (7.7) to keep a volume of sediment of either 500 l or 1 000 l sediment in the scoop (depending of the type of spatula).Transfer
33、 the sediment into the six wells of the two microplates using the tip of the flat spatula to empty the cup of the spatula scoop completely. Each well shall receive 1 000 l sediment (i.e. two scoops of 500 l when using a spatula scoop of 500 l capacity).Proceed similarly with the control sediment (6.
34、6), filling the remaining six wells of the microplates with 1 000 l control sediment.Keeping the microplates horizontal, gently shake them to distribute the sediment evenly over the bottom surface of the wells.Take the container with the algal food suspension (6.4.2), shake it gently to homogenize t
35、he algal suspension, and add 2 ml algal suspension to each well of the two microplates.NOTE Algal food is provided to the organisms at the start of the test to avoid starvation which may lead to mortality or decreased growth of the test organisms.9.5 Transfer of the ostracod neonates into the test c
36、ontainerPut the hatching Petri dish with the neonates on the stage of the stereomicroscope.Fill a small Petri dish (7.2) with 10 ml test medium (6.3) and transfer with the glass micropipette about 65 to 70 ostracods into the Petri dish.Put the Petri dish with the ostracods under the stereomicroscope
37、 and transfer 10 neonates into each well of the first microplate. ISO 2012 All rights reserved 5BS ISO 14371:2012ISO 14371:2012(E)Repeat this transfer operation for the second microplate.On completion of the transfers, cover the two microplates with a sheet, e.g. of polyethylene, and the microplate
38、cover.9.6 Incubation of the test systemIncubate the two microplates at (25 1) C in darkness for 6 d.9.7 Measurements9.7.1 GeneralAt the end of the exposure period, the ostracods have to be recovered from the test containers to determine the percentage mortality and to make the length measurements of
39、 the surviving ostracods.The ostracods can be recovered “directly” from the wells containing control sediment since they are easily visible under a stereomicroscope. This is, however, mostly not the case for the test wells containing test sediment, for which a “sieving procedure” has to be applied t
40、o recover the ostracods.9.7.2 Recovery of the living ostracods from the wells with control sedimentPlace one multiwell on the stage of the stereomicroscope and switch on (preferably) both the top and the bottom illumination.Centre one well containing control sediment and, with the aid of the glass m
41、icropipette, pick up the living ostracods one by one and transfer them into a small Petri dish (7.2) containing test medium (6.3)Perform the same operation for all the wells containing control sediment.9.7.3 Recovery of the ostracods from the wells with test sedimentTake one multiwell, and with a “l
42、arge mouth” pipette (7.5) gently mix the sediment in the first well which contains test sediment, with the layer of test medium (6.3) on top of the sediment.Aspirate part of the sediment suspension, transfer it into the microsieve (7.8) and gently rinse the contents of the microsieve with tap water
43、from a wash bottle until all the fine sediment particles are washed out.Proceed further with the stepwise transfer of the sediment suspension from the well to the microsieve, followed by rinsing with tap water, until most of the sediment has been transferred from the well to the microsieve.Add a few
44、 millilitres of test medium (6.3) to the well, mix it with the remaining part of the sediment, and transfer it to the microsieve for rinsing. Repeat this operation several times, if necessary, to make sure that all the sediment and all the ostracods have been transferred.Turn the microsieve upside d
45、own on top of a small Petri dish (7.2) and rinse the contents of the microsieve back into the Petri dish with test medium (6.3). Make sure that the full contents of the microsieve are transferred into the Petri dish.These transfer and rinsing operations have, subsequently, to be performed for all th
46、e wells with test sediment of the two microplates.NOTE The Petri dishes will contain the remaining (large) sediment particles and the living and dead ostracods which can now easily be seen under a stereomicroscope.The user can, if desired, also apply other techniques to recover the ostracods.9.7.4 S
47、coring of the mortalityPut, one by one, all the Petri dishes containing the ostracods on the stage of the stereomicroscope and count the number of living ostracods in each Petri dish.6 ISO 2012 All rights reservedBS ISO 14371:2012ISO 14371:2012(E)Score the number, nA, of living ostracods counted in
48、each Petri dish on data report template 2 (Table 2) in the appropriate cells of the “Number of living ostracods” row.Subtract each of the scored numbers, nA, from 10 ( i.e. the number of test organisms inoculated in the well at the start) to obtain the number of dead ostracods, 10 - nA, and score th
49、ese numbers on data report template 2 in the cells of the “Number of dead ostracods” row.NOTE The Petri dishes originating from the wells with test sediment can also contain “dead” ostracods. The total number of ostracods (i.e. dead plus living) can still be less than 10. The explanation for this is that, except for manipulation errors during the transfers and the sievings, dead ostracods decompose very rapidly, leaving no visible remains.Proceed the same way with all the Petri dishes containing the test o
