1、Designation: E 1924 97 (Reapproved 2004)Standard Guide forConducting Toxicity Tests with BioluminescentDinoflagellates1,2This standard is issued under the fixed designation E 1924; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, th
2、e year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers two distinct procedures, based onsimilar principles, for obtaining data concerning the ad
3、verseeffects of a test material (added to dilution water) on oceanicbioluminescent dinoflagellates.1.1.1 The endpoint for both procedures is based on ameasurable reduction or inhibition in light output from thedinoflagellates. Both procedures are similar in that whenbioluminescent dinoflagellates ar
4、e exposed to toxicants, ameasurable reduction in bioluminescence is observed fromtheir cells following mechanical stimulation when compared tocontrol cells. In the first procedure, cells of the bioluminescentdinoflagellate Gonyaulax polyedra can be tested over a rangeof up to seven days of exposure
5、(or longer) to a toxicant. Thesecond procedure uses another species, Pyrocystis lunula, for a4 h test.1.2 Both procedures can measure the toxic effects of manychemicals, various marine and freshwater effluents, antifoulingcoatings, leachates, and sediments to bioluminescent di-noflagellates (1-5).3C
6、ompounds with low water solubility suchas large organic molecules may be solubilized with methanol,ethanol, and acetone solvents for testing (4) (see Guide E 729).1.3 An IC50 in light output (bioluminescence) is the rec-ommended endpoint (1). However, percent inhibition of biolu-minescence is an app
7、ropriate endpoint in some cases (5).1.4 Other modifications of these procedures might be justi-fied by special needs or circumstances. Although using appro-priate procedures is more important than following prescribedprocedures, results of tests conducted using unusual proceduresare not likely to be
8、 comparable to results of other tests.Comparison of results obtained using modified and unmodifiedversions of these procedures might provide useful informationconcerning new concepts and procedures for conducting acuteand chronic tests.1.5 The values stated in SI units are to be regarded as thestand
9、ard.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenc
10、ed Documents2.1 ASTM Standards:4D 1141 Practice for the Preparation of Substitute OceanWaterD 5196 Guide for Biomedical Grade WaterE 178 Practice for Dealing with Outlying ObservationsE 729 Guide for Conducting Acute Toxicity Tests withFishes, Macroinvertebrates, and AmphibiansE 1192 Guide for Condu
11、cting Acute Toxicity Tests onAqueous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE 1218 Guide for Conducting Static 96-h Toxicity Testswith MicroalgaeE 1733 Guide for Use of Lighting in Laboratory Testing3. Terminology3.1 Definitions: The words “must,”“ should,” “may,
12、” “can,”and “might” have very specific meanings in this guide.3.1.1 canis used to mean is (are) able to.3.1.2 mayis used to mean is (are) allowed to.3.1.3 mightis used to mean could possibly.3.1.4 mustis used to express an absolute requirement, thatis, to state that the test ought to be designed to
13、satisfy thespecified condition, unless the purpose of the test requires adifferent design.1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current editio
14、n approved August 1, 2004. Published August 2004. Orignallyapproved in 1997. Last previous edition approved in 1997 as E 192497.2This standard Guide is a document developed using the consensus mechanismsof ASTM, that provides guidance for the selection of procedures to accomplish aspecific test but
15、which does not stipulate specific procedures.3The boldface numbers given in parentheses refer to a list of references at theend of the text.4For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volum
16、e information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.5 shouldis used to state that the specified condition isrecommended and ought to be met if possible.3.
17、2 Definitions of Terms Specific to This Standard:3.2.1 bioluminescenceproduction of light by living organ-isms due to an enzyme-catalyzed chemical reaction.3.2.2 dark phasethat part of the daily cycle (night) whendinoflagellates are not being exposed to ambient light andproduce the greatest levels o
18、f bioluminescence when stimu-lated.3.2.3 dinoflagellateunicellular, eukaryotic, flagellatedfresh or marine organisms that have photosynthetic and non-photosynthetic species. Dinoflagellates have brownish plastidscontaining chlorophyll a, chlorophyll c, and a mixture ofcarotenoid pigments, including
19、peridinin that is unique to thisphylum.3.3 IC50a statistically or graphically estimated concen-tration of test material that, under specified conditions, isexpected to cause a 50 % inhibition of a biological process(such as growth, reproduction, or bioluminescence) for whichthe data are not dichotom
20、ous.3.4 luxa unit of illumination equal to the direct illumina-tion that is everywhere 1 m from a uniform point source of onecandle intensity or equal to 11mm2 .3.5 Pyrocystis lunula mutanta mutant that produces 30 %greater light than its progenitor.4. Summary of Guide4.1 Experimental DesignA dinofl
21、agellate test intended toallow calculation of an IC50 usually consists of one or morecontrol treatments and a geometric series of at least fiveconcentrations of test material. In the medium or solventcontrol(s), or both, dinoflagellates are exposed to medium towhich no test material has been added.
22、Samples are usuallydiluted from the highest tested concentration through a seriesof dilutions to 6.25 % of the highest tested concentration.Except for the controls and the highest concentration, eachconcentration should be at least 50 % of the next higher oneunless information concerning the concent
23、ration-effect curveindicates that a different dilution factor is more appropriate. Ata dilution factor of 0.5, five properly chosen concentrations area reasonable comprise between cost and the risk of allconcentrations being either too high or too low.4.1.1 The primary focus of the physical and expe
24、rimentaldesign and the statistical analysis of the data is the experimen-tal unit, which is defined as the smallest physical entity towhich treatments can be independently assigned (6). As thenumber of test cuvettes (experimental units) increases, thenumber of degrees of freedom increases, and, ther
25、efore, thewidth of the confidence interval on a point estimate decreasesand the power of an hypothesis test increases.4.1.2 With respect to factors that might affect results withinthe test chamber and the results of the test, all cuvettes in thetest should be treated as similarly as possible. For ex
26、ample,within the test chamber, the temperature affecting each testcuvette should be as similar as possible unless the purpose ofthe test is to study the effect of light or temperature. Prior to thetest replicates are usually arranged into rows. Placement of thecuvettes must be randomized.4.1.3 The m
27、inimum desirable number of test chambers andcell density per treatment should be calculated from theexpected variance among test cuvettes and either the maximumacceptable width of the confidence interval on a point estimateor the minimum difference that is desired to be detectable usinghypothesis te
28、sting (7).Acoefficient of variation of 10 % or lessin light production between cuvettes is desirable, however,reliable toxicity trends can be observed with a coefficient ofvariation as high as 30 %.4.2 SummaryIf the sample has a salinity of less than 33 62 g/Kg (parts-per-thousand), commercial grade
29、 aquarium seasalt may be added directly to the water sample to bring it intothis range. Testing of the dinoflagellates is accomplished byplacing individual cuvettes containing the test material, me-dium, and cells into a darkened test chamber which is attachedto a photomultiplier tube (PMT). The top
30、 of the test chambermust be removable and house a small motor that drives a steelshaft terminating in a propeller. The propeller is seated intoeach cuvette and, as the contents are stirred at a constantvoltage, bioluminescence is generated and measured by thePMT. At the end of each stir period, the
31、accumulated “PMTcounts” are shown on an LED display. Each test period iscompleted at preset intervals thereafter until completion of thetoxicity test. Pyrocystis lunula mutant can be used to conducta 4 h acute test. Gonyaulax polyedra can be used in testsconducted for four to seven days or longer de
32、pending on thepurpose of the test. Mean light output (stimulated biolumines-cence expressed as PMT counts) is calculated for each treat-ment and control. Light output means (as percent of controls)are plotted against time. An IC50 can be estimated for eachdays of the test (1).5. Significance and Use
33、5.1 Protection of aquatic species requires prevention ofunacceptable effects on populations in natural habitats. Toxic-ity tests are conducted to provide data to predict what changesin viable numbers of individual species might result fromsimilar exposure in the natural habit. Information might also
34、 beobtained on the effects of the material on the health of otherspecies. Bioluminescent dinoflagellates represent an importanteucaryotic group which are widely distributed in the oceanicenvironment.6. Hazards6.1 Many materials can affect humans adversely if precau-tions are inadequate. Therefore, s
35、kin contact with all testmaterials, solutions, and leachates should be minimized bysuch means as wearing appropriate protective gloves (espe-cially when washing equipment or putting hands in testsolutions), laboratory coats, aprons, and safety glasses. Infor-mation on toxicity to humans (8), recomme
36、nded handlingprocedures (9), and the chemical and physical properties of thetest material should be studied before a test is begun.6.2 Disposal of stock solutions and test solutions might posespecial problems in some cases. Therefore, health and safetyprecautions and applicable regulations should be
37、 consideredbefore beginning a test.6.3 To prepare dilute acid solutions, concentrated acidshould be added to water, not vice versa. Opening a bottle ofE 1924 97 (2004)2concentrated acid and mixing with water should be performedonly in a well-ventilated area or under a fume hood.7. Laboratory Equipme
38、nt7.1 FacilitiesThe culture trays for the dinoflagellates, amicroscope for estimating the cell stock density, the prepara-tion of test materials, leachate preparation, pipetting of the cellsinto cuvettes, and the testing of the dinoflagellates with anappropriate test chamber-PMT combination. Suffici
39、ent labora-tory counter space should accomodate “wet” preparation of allstock solutions, cell counting, and culture of the dinoflagel-lates. The glassware should be clean rinsed with a high qualitywater such as deionized or distilled. The dinoflagellates mustbe maintained in a temperature incubator
40、of 18 to 20C asabrupt changes in their temperature could effect the viability ofthe cells and their light output. The incubator must be fittedwith cool white fluorescent bulbs (40 watts each) to provideillumination of approximately 1075 lux to Pyrocystis lunulaand 4000 lux to Gonyaulax polyedra (400
41、0 lux is the approxi-mate equivalent of 6 to 10 w/m2; see Guide E 1733) for thegrowth of the photosynthetic dinoflagellates. The light fluenceshould be monitored adjacent to various test chambers at theheight of the surface of the test solutions. Light fluence mustnot deviate by more than 10 % from
42、the desired level. Layersof cheese cloth or screen material may be used to attenuatelight incident upon flasks and test chambers, if necessary. Atimer should be provided to turn the lights on and off in aprescribed 12:12 h (light:dark) cycle. For convenience oftesting in the laboratory, the cells ca
43、n be exposed to lightduring the hours from 2200 to 1000. The cells would then bein their dark phase from 1000 to 2200. The cells are moststimulable 3 to 5 h into their dark phase and consequentlyproduce maximum levels of light (bioluminescence) duringthis period. Cells must be shielded from ambient
44、room lightsduring their dark phase and during testing. A black cloth maybe used for this purpose. The operator may conduct tests in adarkened laboratory with a red light for ease of operation. Thispractice can prevent unnecessary exposure of the test organ-isms to light, which could cause unpredicta
45、ble biolumines-cence.7.2 Culture and Test ChambersOptical grade disposablespectrophotometric cuvettes or clear, borosilicate sample vialsshould be used as test chambers. Cultures should be maintainedin borosilicate Erlenmeyer flasks.All vials and flasks should beseawater-aged for several days prior
46、to first time use. Dispos-able cuvettes should be soaked in deionized water for severalhours prior to use in a test. Disposable cuvettes should bediscarded following the test.7.3 Bioluminescence Measurement SystemOne possibleconfiguration for a toxicity test system uses a 2-in. diameterRCA 8575 phot
47、omultiplier tube (PMT) with an S-20 response(300 to 820 nm; peak sensitivity 428 nm) (2). Another systemuses a 931B or H957-06 miniature PMT (5). The top of the testchamber must be removable and house a small adjustablemotor which drives a stainless steel shaft terminating in aplastic propeller. The
48、 propeller is seated into the cuvette and, asthe contents are stirred, bioluminescence is generated andmeasured by the PMT. At the end of each stir period, theaccumulated PMT counts are shown in an LED display. Eachtest period is conducted at preset intervals (either at 4 or 24 h)until completion of
49、 the toxicity test.8. Medium8.1 Either synthetic seawater or seawater that is enriched areappropriate media for culture and dilution purposes. Naturalseawater by itself is not adequate to maintain high densities ofdinoflagellates in culture. Seawater with added nutrients (En-riched Seawater Medium-ESM; see Guide E 1218) or Syn-thetic Dinoflagellate Medium (SDM) (10) are used to ensuregrowth in control replicates and cultures. Either ESM or SDMis recommended for use in these tests.8.1.1 Purity of ReagentsReagent grade chemicals shouldbe used for the preparation of en
