1、Designation: E1498 92 (Reapproved 2012)Standard Guide forConducting Sexual Reproduction Tests with Seaweeds1This standard is issued under the fixed designation E1498; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last
2、 revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers procedures for obtaining laboratorydata concerning the adverse effects of a test material added todiluti
3、on water on sexual reproduction by seaweeds. Theexposure duration is species dependent and is followed by aperiod of development to allow the evidence of sexual repro-duction to appear. There is no exposure to toxicants during thedevelopment period. This restricts the tests primarily to theevents su
4、rrounding egg fertilization, and it minimizes anytimelag effects on development that might interfere withcorrect enumeration of the number of sexual events thatoccurred. These procedures will probably be useful for con-ducting sexual reproduction toxicity tests with a variety ofspecies of seaweeds,
5、although modifications might be neces-sary.1.2 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, the results of tests conducted using unusual pro-cedures are not
6、 likely to be comparable to those of many othertests. Comparison of the results obtained using modified andunmodified versions of these procedures might provide usefulinformation concerning new concepts and procedures forconducting sexual reproduction tests with seaweeds.1.3 These procedures are app
7、licable to most chemicals,either individually or in formulations, commercial products,and known mixtures or whole effluents, as well as for use intesting surface waters. With appropriate modifications, theseprocedures can be used to study the effects of temperature,dissolved oxygen, pH, and such mat
8、erials as leachates, oils,particulate matter, and sediments.1.4 The values stated in SI units are to be regarded as thestandard.1.5 This guide is arranged as follows:SectionReferenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Apparatus 6Facilities 6.1Construction Materials 6.2T
9、est Chambers 6.3Cleaning 6.4Acceptability 6.5Hazards 7Dilution and Culture Water 8Requirements 8.1Source and Treatment 8.2Test Material 9Single Chemicals 9.1Stock Solutions 9.2Effluents and Surface Waters 9.3Test Concentrations 9.4Test Organism 10Species 10.1Life Stage 10.2Source 10.3Culture Nutrien
10、t Medium 10.4Procedure 11Preparation of Plants for a Test 11.1Test Conditions 11.2Experimental Design 11.3Acceptability of Test 12Calculation of Results 13Documentation 14Keywords 15Appendix X11.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. I
11、t 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. For specific hazardsstatements, see 6.4 and Section 7.2. Referenced Documents2.1 ASTM Standards:2E380 Practice for Use of
12、 the International System of Units(SI) (the Modernized Metric System) (Withdrawn 1997)3E729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-ians1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fa
13、teand is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved Dec. 1, 2012. Published January 2013. Originallyapproved in 1992. Last previous edition approved in 2004 as E1498 92 (2004).DOI: 10.1520/E1498-92R12.2For referenced ASTM standards,
14、visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright
15、 ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1E943 Terminology Relating to Biological Effects and Envi-ronmental FateE1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE1192 Guide for Conducting Acute Toxici
16、ty Tests on Aque-ous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and Amphibians3. Terminology3.1 Definitions:3.1.1 The words “must,” “should,” “may,” “can,” and“might” have very specific meanings in this guide. “Must” isused to express an absolute requirement, that is, to state tha
17、t thetest ought to be designed to satisfy the specified condition,unless the purpose of the test requires a different design.“Must” is used only in connection with factors that relatedirectly to the acceptability of the test (see Section 12).“Should” is used to state that the specified condition isr
18、ecommended and ought to be met, if possible. Although theviolation of one “should” is rarely a serious matter, theviolation of several will often render the results questionable.Terms such as “is desirable,” “is often desirable,” and “mightbe desirable” are used in connection with less importantfact
19、ors. “May” is used to mean “is (are) allowed to,” “can” isused to mean “is (are) able to,” and “might” is used to mean“could possibly.” Therefore, the classic distinction betweenmay and can is preserved, and might is never used as asynonym for either “may” or “can.”3.1.2 For definitions of other ter
20、ms used in this guide, referto Guide E729, Terminology E943, and Guide E1023. For anexplanation of units and symbols, refer to Practice E380.3.2 Definitions of Terms Specific to This Standard:3.2.1 cystocarpa structure produced by the female redalgal gametophyte in response to fertilization.3.2.2 ga
21、metophytethe sexual, gamete-producing phase inthe life history of a plant.3.2.3 ostiolean opening.3.2.4 sorus (plural sori)a group or cluster of reproductivestructures, for example, spermatangia, producing male gam-etes.3.2.5 spermatiamale gametes in red algae; non-motile andcolorless.3.2.6 trichogy
22、nean elongation of a female oogonium (eggcell) to which male gametes become attached.4. Summary of Guide4.1 In each of two or more treatments, female and malegametophytes are exposed in each of three or more testchambers for two days under static or renewal conditions. Ineach of one or more control
23、treatments, the gametophytes aremaintained in dilution water to which no test material has beenadded, in order to provide the following: (1) a measure of theacceptability of the test by giving an indication of the qualityof the plants and suitability of the dilution water, testconditions, and handli
24、ng procedures; and (2) the basis forinterpreting data obtained from the other treatments. In each ofone or more treatments, the gametophytes are maintained indilution water to which a selected concentration of test materialhas been added. At the end of the exposure period, femalegametophytes are rem
25、oved and incubated (if necessary) for anadditional period of time in toxicant-free medium to allow thedevelopment of structures created from sexual reproduction(that is, germination of the zygote). At the end of thedevelopment period, sexually produced structures are counted,and the number in the co
26、ntrols is compared with those in thetreatments to determine the effect of the test material. Theresults can be reported as either IC50 or No Observed EffectConcentration (NOEC) and Lowest Observed Effect Concen-tration (LOEC) based on sexual reproduction.5. Significance and Use5.1 Seaweeds have hist
27、orically been considered less usefulfor toxicity testing than microalgae (1),4and microalgae areoften considered less sensitive than aquatic animals (2). Suchconclusions concerning seaweed insensitivity were based ondata for only a few hardy species and based generally onvegetative growth of adult s
28、tages as the primary endpoint. Thesensitivity of seaweeds increases when effects on sexualreproduction are assessed. This has been shown for Champiaparvula(3), as well as for the brown seaweeds, Fucusedentatus, Laminaria saccharina, and Macrocystis pyrifera(4).5.2 The results of sexual reproduction
29、tests with seaweedsmight be useful for predicting the long-term effects likely tooccur on seaweeds in field situations due to exposure undercomparable conditions.5.3 The results of sexual reproduction tests with seaweedsmight be used to compare the chronic toxicities of differentmaterials, and also
30、to study the effects of various environmen-tal factors on the results of such tests.5.4 The results of sexual reproduction tests with seaweedsmight be an important consideration when assessing thehazards of materials to aquatic organisms or when derivingwater quality criteria for saltwater organisms
31、 (5).5.5 The results of sexual reproduction tests with seaweedswill depend on the temperature, composition of dilution water,condition of test organisms, and other factors such as light andmedia.6. Apparatus6.1 FacilitiesStock cultures and test chambers should bemaintained in constant-temperature in
32、cubators or water baths.The air used for aeration should be free of fumes, oil, andwater; filters to remove oil and water are desirable. Filtration ofthe air through a 0.22-m bacterial filter might be desirable tohelp prevent the contamination of stock cultures with air-bornemicroalgae. To further r
33、educe the possibility of contaminationby test materials and other substances, especially volatile ones,the stock cultures ought not be in a room in which toxicity testsare conducted, stock solutions or test solutions are prepared, or4The boldface numbers in parentheses refer to the list of reference
34、s at the endof this guide.E1498 92 (2012)2equipment is cleaned. A timing device should be used toprovide a 16-h light and 8-h dark photoperiod.6.2 Construction MaterialsEquipment and facilities thatcontact the stock solutions, test solutions, or any water intowhich the test organisms will be placed
35、should not containsubstances that can be leached or dissolved by aqueoussolutions in amounts that affect the growth or reproduction ofseaweeds adversely. In addition, equipment and facilities thatcontact the stock solutions or test solutions should be chosen tominimize the sorption of test materials
36、 from water. Glass ispreferred and should be used whenever possible, althoughsome brands of polycarbonate and polystyrene have been usedwith success. Brass, copper, lead, galvanized metal, and naturalrubber should not contact dilution water, stock solutions, or testsolutions before or during the tes
37、t. Items made of neoprenerubber, or other materials not mentioned previously, should notbe used unless it has been shown that their use will not affectthe survival, growth, or reproduction of seaweeds adversely.6.3 Test Chambers:6.3.1 In a toxicity test with aquatic organisms, test chambersare defin
38、ed as the smallest physical units between which thereare no water connections. The chambers should be covered tokeep out extraneous contaminants and to reduce the evapora-tion of test solution and test material. The cover should betransparent to minimize shading. All chambers, covers, andcompartment
39、s in a test should be identical.6.3.2 The volume used for exposure of the test organisms isspecies dependent. The use of excessively large volumes ofsolution in the test chambers will probably increase unneces-sarily the amount of dilution water and test material used.Glass, 125-mL Erlenmeyer flasks
40、, and 200-mL polystyreneparty cups have been used successfully.6.4 CleaningAll culture glassware should be acid-strippedin 15 % HCl after being washed with a phosphate-free deter-gent by soaking in 15 % concentrated HCl for at least 10 min(or with three successive rinses with dilute acid coating the
41、entire interior surface). Following the acid treatment, rinsethree times in deionized water. The acid treatment is necessarybecause some detergents can leave a residue that is toxic toseaweeds. Culture glassware should be baked periodically (atleast every six months) in a muffle furnace to remove th
42、eorganic material that might build up on its surface.Alternately,a few mLof concentrated sulfuric acid can be rolled around theinside of wet glassware. Caution: The addition of acid to thewet glassware generates heat.6.4.1 At the end of a test, all items that will be used againshould immediately be
43、(1) emptied; (2) rinsed with water (tapwater is permissible at this stage); (3) cleaned by a procedureappropriate for removing the test material (for example, acid toremove metals and bases; detergent, organic solvent, or acti-vated carbon to remove organic chemicals); and (4) cleaned asabove for cu
44、lture glassware.6.5 AcceptabilityBefore sexual reproduction tests withseaweeds are conducted in a new test facility or with newequipment, it is desirable to conduct a nontoxicant test, inwhich all test chambers contain dilution water with no addedtest material. This will determine the following befo
45、re the firsttest: (1) whether the species selected to be used reproducesacceptably; (2) whether the water and culture and handlingprocedures are acceptable; (3) whether there are any locationeffects on reproduction; and (4) the magnitude of the withinchamber and between-chamber variances.7. Hazards7
46、.1 Many materials can affect humans adversely if theprecautions taken are inadequate. Skin contact with all testmaterials and solutions should therefore be minimized bywearing appropriate protective gloves (especially when wash-ing equipment or putting hands into test solution), laboratorycoats, apr
47、ons, and glasses, and by using forceps to removeseaweeds from test solutions. Special precautions, such ascovering the test chambers and ventilating the area surroundingthe chambers, should be taken when conducting tests onvolatile materials. Information on toxicity to humans (6),recommended handlin
48、g procedures (7), and chemical andphysical properties of the test materials should be studiedbefore a test is begun. Special procedures might be necessarywith radiolabeled materials (8) and with test materials that are,or are suspected of being, carcinogenic (9).7.2 Although the disposal of stock so
49、lutions, test solutions,and test organisms poses no special problems in most cases,health and safety precautions and applicable regulations shouldbe considered before beginning a test. The removal or degra-dation of test material might be desirable before the disposal ofstock and test solutions.7.3 The cleaning of equipment with a volatile solvent, suchas acetone, should be performed under a fume hood within aroom in which no smoking is allowed and no open flame, suchas a pilot light, is present.7.4 An acidic solution should not be mixed with a hypochlo-rite solution beca
