1、 Reference number ISO/TR 11044:2008(E) ISO 2008TECHNICAL 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 ISO/TR 1
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6、dress below or ISOs member body in the country of the requester. 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 reservedISO/TR 11044:2008(E) ISO 2008 All right
7、s reserved iii Contents Page Foreword iv Introduction v 1 Scope 1 2 Normative references 1 3 Terms and definitions .1 4 General 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 costatu
8、m9 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 Incubation conditions .21 6.5 Test endpoint .23 Bibliography 25 ISO/TR 11044:2008(E) iv ISO 2008 All rights reservedForeword ISO (the International Organization for Standardiz
9、ation) 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 right to be
10、 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. International Standar
11、ds 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 voting. Publication as an Intern
12、ational 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 that which is normally published as an International Standard (“state of the art”, for example), it may decide
13、 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 provides are considered to be no longer valid or useful. Attention is drawn to the possibility that some of t
14、he 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 Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. ISO/TR 11044:2008(E) ISO 2008 All righ
15、ts 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). The characteristics of these phases are depe
16、ndent 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 medium. The first systematic application of
17、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 was used to assess fertilizing influences o
18、f 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 in southeast Norway in 1959. It has become t
19、he 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 was early recognized that bioassays of microa
20、lgae 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 some authors claimed that the sensitivity of
21、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 the other hand, microalgae are generally th
22、e 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 undertaken to increase the reproducibility and
23、 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 sensitive of the “base-set” tests which include a
24、lso 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 guidelines and standards for growth inhibition
25、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 series of documents were developed in parallel
26、 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 was produced in 1982, but the first ISO/DIS
27、 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 compounds, metals and waste water, was revised as I
28、SO 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. TECHNICAL REPORT ISO/TR 11044:2008(E) ISO 2008
29、 All rights reserved 1 Water quality Scientific and technical aspects of batch algae 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 specifie
30、d in ISO 8692, for freshwater, and ISO 10253, for marine waters. Previously unpublished 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
31、 for the application of this document. For dated references, only the edition cited applies. 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
32、 10253:2006, Water quality Marine algal growth inhibition test with Skeletonema costatum 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 purpo
33、ses of this document, the following terms and definitions apply. 3.1 effective concentration EC xconcentration 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 EC xis used in this Technical Report
34、to mean E r C xwhere “r” denotes “rate”. Effective concentrations based on area under the growth curve can be derived, and these are designated E b C x , 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
35、: 1d d n nt = ISO/TR 11044:2008(E) 2 ISO 2008 All rights reservedwhere n is the cell density, expressed in cells per millilitre; t is the time, expressed 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 inhib
36、ition 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 an exponentially growing inoculum culture and incubated under continuous illumination. The growth medium, inoculum biomass density, temperature, a
37、nd illuminance, have been selected to allow an exponential increase in the algal biomass density during the 72 h incubation period for the recommended test species. The experimental design of the tests includes a series of five or more concentrations of the test material in growth medium prepared in
38、 triplicate, and six control replicates without test material. After inoculation with test algae, the solutions are incubated in transparent, inert containers under continuous illumination and constant temperature. The cultures should be agitated in order to obtain a homogenous suspension of the alg
39、ae 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 until termination of the test after 72 h. An example of a growth inhibition test with Pseudokirchneriella subcapitata is shown in Figure 1. The substa
40、nce tested was potassium dichromate. The growth curves show close adherence to exponential growth in the cultures, and decreasing growth rates with increasing concentration of the test substance. Average specific growth rates may be calculated as the logarithmic increase in cell density from start t
41、o 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 using a log-logistic model (REGTOX) 1) . Concentrations causing 10 % and 50 % reduction of the growth rate (EC 10and EC 50respectively) have been
42、calculated from the regression equation. 1) Available (2008-11-14) at http:/eric.vindimian.9online.fr/ ISO/TR 11044:2008(E) ISO 2008 All rights reserved 3Key 1 (K 2 Cr 2 O 7 ) = 0 (control) 5 (K 2 Cr 2 O 7 ) = 1 mg/l 2 (K 2 Cr 2 O 7 ) = 0,25 mg/l 6 (K 2 Cr 2 O 7 ) = 1,6 mg/l 3 (K 2 Cr 2 O 7 ) = 0,4
43、mg/l n cell density, 10 3cells/ml 4 (K 2 Cr 2 O 7 ) = 0,63 mg/l t time, h Figure 1 Growth curves (mean values of replicates) for cultures of P. subcapitata at different mass concentrations of K 2 Cr 2 O 7ISO/TR 11044:2008(E) 4 ISO 2008 All rights reservedKey specific growth rate as a percentage of c
44、ontrol EC 10effective concentration at 10 % inhibition EC 50effective concentration at 50 % inhibition (K 2 Cr 2 O 7 ) potassium dichromate mass concentration, mg/l Figure 2 Mass concentration/response plot showing the effect of K 2 Cr 2 O 7on the growth rate of P. subcapitata 5 Test species 5.1 Gen
45、eral Microalgae constitute a phylogenetically diverse group of organisms, including the procaryotic cyanobacteria and several phyla of eucaryotic algae. It is therefore not surprising that the sensitivity among different species of microalgae to various toxic substances is highly variable. Some stud
46、ies have shown that such interspecies variation in sensitivity may amount to three to four orders of magnitude (References 2, 24, 54). This variation in sensitivity must, of course, be acknowledged when intrepreting data on algal toxicity in a risk assessment context and the use of a battery of spec
47、ies has been proposed to account for the variation (References 8, 21, 33, 53). ISO 8692 specifies two green algae P. subcapitata and Desmodesmus subspicatus (previously known as Scenedesmus subspicatus) as test species in freshwater. ISO 10253 specifies two marine diatoms, Skeletonema costatum and P
48、haeodactylum tricornutum for the marine algae growth inhibition test. A search for data entries on toxicity of chemicals to the algal species included in the ISO and OECD test methods in the US EPA database ECOTOX showed a total of approximately 5 000 data entries of which 42 % are from tests with P
49、. subcapitata, which confirms the position of this strain as a reference alga in bioassays (see Figure 3). Among the marine species, S. costatum appears to be the one most frequently used. ISO/TR 11044:2008(E) ISO 2008 All rights reserved 5Key 1 P. subcapitata 5 Navicula pelliculosa 2 Chlorella vulgaris 6 S. costatum 3 D. subspicatus 7 P. tricornutum 4 Anabaena flos-aquae n enumber of entries aFreshwater algae. bMarine algae. Figure 3 Number of data entries on toxicity to algae
