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1、 Reference number ISO/TR 11044:2008(E) ISO 2008 TECHNICAL REPORT ISO/TR 11044 First edition 2008-12-01 Water quality Scientific and technical aspects of batch algae growth inhibition tests Qualit de leau Aspects scientifiques et techniques des essais dinhibition de croissance dun lot dalgues Copyrig
2、ht International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 01/31/2009 01:56:38 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO/TR 11044:2008(E) PDF disclaimer This PDF file may contain embedded
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6、d. 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 below or ISOs member body in the country of the requester
7、. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2008 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licen
8、see=Boeing Co/5910770001 Not for Resale, 01/31/2009 01:56:38 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO/TR 11044:2008(E) ISO 2008 All rights reserved iii Contents Page Foreword iv Introduction v 1 Scope 1 2 Normative references1 3 Terms and definitions .1 4 General
9、 principles of ISO algal growth inhibition tests.2 5 Test species .4 5.1 General4 5.2 Pseudokirchneriella subcapitata6 5.3 Desmodesmus subspicatus.9 5.4 Skeletonema costatum9 5.5 Phaeodactylum tricornutum.12 6 Test conditions 14 6.1 Growth medium14 6.2 pH control.16 6.3 Inoculum density .18 6.4 Incu
10、bation conditions .21 6.5 Test endpoint .23 Bibliography25 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 01/31/2009 01:56:38 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO/T
11、R 11044:2008(E) iv ISO 2008 All rights reserved Foreword ISO (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 ISO technical committees. Each m
12、ember 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 closely with the International
13、Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. 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 a
14、dopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. In exceptional circumstances, when a technical committee has collected data of a different kind from tha
15、t which is normally published as an International Standard (“state of the art”, for example), it may decide by a simple majority vote of its participating members to publish a Technical Report. A Technical Report is entirely informative in nature and does not have to be reviewed until the data it pr
16、ovides are considered to be no longer valid or useful. 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. ISO/TR 11044 was prepared by Technical Committ
17、ee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 01/31/2009 01:56:38 MSTNo reproduction or networking permitted without license from IHS -
18、,-,- ISO/TR 11044:2008(E) ISO 2008 All rights reserved v Introduction The growth of microalgae in batch cultures follows a well known pattern, with a lag phase followed by an exponential growth phase, a phase of declining growth rate, a stationary phase, and ultimately a death phase (Reference 9). T
19、he characteristics of these phases are dependent on the environmental conditions including the chemical composition of the growth medium, which provides the basis for using batch cultures of algae as bioassays to investigate growth stimulating or inhibiting properties of constituents of the growth m
20、edium. The first systematic application of microalgae bioassays for which standard protocols were developed was for assessment of nutrient status and identification of growth limiting nutrients. Skulberg (Reference 50) developed a bioassay with the green alga Selenastrum capricornutum Printz, which
21、was used to assess fertilizing influences of pollution in inland waters. The nutrient bioassay with S. capricornutum was further developed and standardised in Reference 55. The strain of S. capricornutum used as test organism in the nutrient bioassays was originally isolated from the river Nitelva i
22、n southeast Norway in 1959. It has become the most commonly used test algae for bioassays and is available from most major culture collections. Due to taxonomic revisions, it was first renamed Raphidocelis subcapitata and later Pseudokirchneriella subcapitata (Korshikov) Hindak (Reference 20). It wa
23、s early recognized that bioassays of microalgae could be used to study the growth-inhibiting effects of toxic chemicals and waste waters, and a modification of the algal assay procedure for toxicity studies was made in Reference 43. However, based on compilations of early algae toxicity test data so
24、me authors claimed that the sensitivity of algae generally was low (Reference 26). The environmental relevance of results of the tests was also questioned because of the significant interspecies variation in response and lack of field-validation of results of algal toxicity tests (Reference 28). On
25、the other hand, microalgae are generally the most important primary producers in aquatic ecosystems. Excluding the assessment of toxicity to this group of organisms in risk assessment and environmental management cannot be justified. Development and standardisation of methods have therefore been und
26、ertaken to increase the reproducibility and relevance of toxicity tests with microalgae. Standardised growth inhibition tests with algae are now a cornerstone in the environmental management and risk assessment of chemicals. Recent reviews (e.g. Reference 57) show that they are often the most sensit
27、ive of the “base-set” tests which include also acute toxicity tests with fish and Daphnia. In addition to several national organisations, the Organisation for Economic Co-operation and Development (OECD) and the International Organization for Standardization (ISO) took on the work of developing guid
28、elines and standards for growth inhibition with microalgae in the late 1970s. The OECD guidelines aim to test chemical substances, while ISO documents cover tests for composite water samples, such as waste water and elutriates. However, harmonisation of the procedures was an objective as the two ser
29、ies of documents were developed in parallel by the two organisations. The development of the freshwater test was initiated by ISO in 1978. Three ring tests were organised between 1980 and 1982 and included in ISO 8692:1989, revised as ISO 8692:2004. The first draft of a marine algae inhibition test
30、was produced in 1982, but the first ISO/DIS was not published until 1991, when the method had been ring tested. ISO 10253:1995 was revised as ISO 10253:2006. In addition to these two standards, ISO 14442:1999, guidelines for algal growth inhibition tests with poorly soluble matter, volatile compound
31、s, metals and waste water, was revised as ISO 14442:2006. In this Technical Report, the general principles of the batch culture growth inhibition tests, and how some critical methological aspects have been addressed in the International Standards for algal growth inhibition tests, are presented. Cop
32、yright International Organization for Standardization Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 01/31/2009 01:56:38 MSTNo reproduction or networking permitted without license from IHS -,-,- Copyright International Organization for Standardization Provided b
33、y IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 01/31/2009 01:56:38 MSTNo reproduction or networking permitted without license from IHS -,-,- TECHNICAL REPORT ISO/TR 11044:2008(E) ISO 2008 All rights reserved 1 Water quality Scientific and technical aspects of batch algae
34、growth inhibition tests 1 Scope This Technical Report discusses scientific and technical aspects that have been considered in connection with the development of batch algal growth inhibition test procedures specified in ISO 8692, for freshwater, and ISO 10253, for marine waters. Previously unpublish
35、ed results of experiments performed at the Norwegian Institute for Water Research (NIVA) have been included to demonstrate various aspects. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited a
36、pplies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 8692:2004, Water quality Freshwater algal growth inhibition test with unicellular green algae ISO 10253:2006, Water quality Marine algal growth inhibition test with Skeletonema costa
37、tum and Phaeodactylum tricornutum ISO 14442, Water quality Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 effective concen
38、tration ECx concentration of test sample which results in a reduction of x % in the specific growth rate relative to the controls ISO 8692 NOTE Unless otherwise stated, the form ECx is used in this Technical Report to mean ErCx where “r” denotes “rate”. Effective concentrations based on area under t
39、he growth curve can be derived, and these are designated EbCx, where “b” denotes “biomass” (see 6.5 for further details). 3.2 specific growth rate proportional rate of increase in cell density per unit of time: 1 d d n nt = Copyright International Organization for Standardization Provided by IHS und
40、er license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 01/31/2009 01:56:38 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO/TR 11044:2008(E) 2 ISO 2008 All rights reserved where n is the cell density, expressed in cells per millilitre; t is the time, expressed
41、 in days. NOTE 1 Specific growth rate is expressed in reciprocal days. NOTE 2 Adapted from ISO 8692. 4 General principles of ISO algal growth inhibition tests The algae growth inhibition test methods specified in ISO 8692 and ISO 10253 are based on batch cultures which are inoculated with algae from
42、 an exponentially growing inoculum culture and incubated under continuous illumination. The growth medium, inoculum biomass density, temperature, and illuminance, have been selected to allow an exponential increase in the algal biomass density during the 72 h incubation period for the recommended te
43、st species. The experimental design of the tests includes a series of five or more concentrations of the test material in growth medium prepared in triplicate, and six control replicates without test material. After inoculation with test algae, the solutions are incubated in transparent, inert conta
44、iners under continuous illumination and constant temperature. The cultures should be agitated in order to obtain a homogenous suspension of the algae and to stimulate gas exchange with the atmosphere. The biomass density in the cultures is measured by direct or indirect methods at 24 h intervals unt
45、il termination of the test after 72 h. An example of a growth inhibition test with Pseudokirchneriella subcapitata is shown in Figure 1. The substance tested was potassium dichromate. The growth curves show close adherence to exponential growth in the cultures, and decreasing growth rates with incre
46、asing concentration of the test substance. Average specific growth rates may be calculated as the logarithmic increase in cell density from start to 72 h. Figure 2 shows the concentration/response plot for the endpoint growth rate. A curve has been fitted to the observations by non-linear regression
47、 using a log-logistic model (REGTOX) 1). Concentrations causing 10 % and 50 % reduction of the growth rate (EC10 and EC50 respectively) have been calculated from the regression equation. 1) Available (2008-11-14) at http:/eric.vindimian.9online.fr/ Copyright International Organization for Standardiz
48、ation Provided by IHS under license with ISO Licensee=Boeing Co/5910770001 Not for Resale, 01/31/2009 01:56:38 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO/TR 11044:2008(E) ISO 2008 All rights reserved 3 Key 1 (K2Cr2O7) = 0 (control) 5 (K2Cr2O7) = 1 mg/l 2 (K2Cr2O7) = 0,25 mg/l 6 (K2Cr2O7) = 1,6 mg/l 3 (K2Cr2O7) = 0,4 mg/l n cell density, 103 cells/ml 4 (K2Cr2O7) = 0,63 mg/l t time, h Figure 1 Growth curves (mean values of