1、Designation: E 1498 92 (Reapproved 2004)Standard Guide forConducting Sexual Reproduction Tests with Seaweeds1This standard is issued under the fixed designation E 1498; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of la
2、st 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 procedures for obtaining laboratorydata concerning the adverse effects of a test material added todil
3、ution 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
4、 surrounding 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 seaweed
5、s, 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
6、not 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
7、applicable 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
8、materials 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
9、.2Test 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 Nutr
10、ient 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
11、. 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. For specific hazardsstatements, see 6.4 and Section 7.2. Referenced Documents2.1 ASTM Standards:2E 380 Practice for Us
12、e of the International System of Units(SI) (the Modernized Metric System)3E 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE 943 Terminology Relating to Biological Effects and En-vironmental Fate1This guide is under the jurisdiction of
13、ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published April 2004. Originallyapproved in 1992. Last previous edition approved in 1998 as E 1498 92 (19
14、98).2For referenced ASTM standards, 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.3Withdrawn.1Copyright ASTM International, 100 Barr Harbor
15、 Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.E 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1192 Guide for Conducting Acute Toxicity Test on Aque-ous Ambient Samples with Effluents with Fishes, Macro-invertebrates, and Amphibians3. Ter
16、minology3.1 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 that thetest ought to be designed to satisfy the specified condition,unless the purpose of the test requir
17、es 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 isrecommended and ought to be met, if possible. Although theviolation of one “should” is rarely a serious
18、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 importantfactors. “May” is used to mean “is (are) allowed to,” “can” isused to mean “is (are) able to,” and “might”
19、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 terms used in this guide, referto Guide E 729, Terminology E 943, and Guide E 1023. For anexplanation of u
20、nits and symbols, refer to Practice E 380.3.2 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 gametophytethe sexual, gamete-producing phase inthe life history of a plant.3.2.3 ostiolean opening.3
21、.2.4 sorus (plural sori)a group or cluster of reproductivestructures, for example, spermatangia, producing male ga-metes.3.2.5 spermatiamale gametes in red algae; non-motileand colorless.3.2.6 trichogynean elongation of a female oogonium (eggcell) to which male gametes become attached.4. Summary of
22、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 treatments, the gametophytes aremaintained in dilution water to which no test material has beenadde
23、d, 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, test condi-tions, and handling procedures; and (2) the basis for interpret-ing data obtained from the other treatments. In ea
24、ch of one ormore treatments, the gametophytes are maintained in dilutionwater to which a selected concentration of test material hasbeen added. At the end of the exposure period, femalegametophytes are removed and incubated (if necessary) for anadditional period of time in toxicant-free medium to al
25、low 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 controls is compared with those in thetreatments to determine the effect of the test material. Th
26、eresults 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 historically been considered less usefulfor toxicity testing than microalgae (1),4and microalgae ar
27、eoften 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 stages as the primary endpoint. Thesensitivity of seaweeds increases when effects on sexualrepro
28、duction are assessed. This has been shown for Champiaparvula(3), as well as for the brown seaweeds, Fucus edenta-tus, Laminaria saccharina, and Macrocystis pyrifera (4).5.2 The results of sexual reproduction tests with seaweedsmight be useful for predicting the long-term effects likely tooccur on se
29、aweeds 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 to study the effects of various environmen-tal factors on the results of such tests.5.4 The
30、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 (5).5.5 The results of sexual reproduction tests with seaweedswill depend on the temperatur
31、e, 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 incubators or water baths.The air used for aeration should be free of fumes, oil, andwater; fi
32、lters 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 reduce the possibility of contaminationby test materials and other substances, especially vol
33、atile ones,the stock cultures ought not be in a room in which toxicity testsare conducted, stock solutions or test solutions are prepared, orequipment is cleaned. A timing device should be used toprovide a 16-h light and 8-h dark photoperiod.6.2 Construction MaterialsEquipment and facilities thatcon
34、tact the stock solutions, test solutions, or any water intowhich the test organisms will be placed should not containsubstances that can be leached or dissolved by aqueoussolutions in amounts that affect the growth or reproduction of4The boldface numbers in parentheses refer to the list of reference
35、s at the endof this guide.E 1498 92 (2004)2seaweeds adversely. In addition, equipment and facilities thatcontact the stock solutions or test solutions should be chosen tominimize the sorption of test materials from water. Glass ispreferred and should be used whenever possible, althoughsome brands of
36、 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 test. Items made of neoprenerubber, or other materials not mentioned previously, should notbe
37、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 defined as the smallest physical units between which thereare no water connections. The chambers
38、 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, andcompartments in a test should be identical.6.3.2 The volume used for exposure of the test organisms is
39、species 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, and 200-mL polystyreneparty cups have been used successfully.6.4 CleaningAll culture glas
40、sware should be acid-stripped in 15 % HCl after being washed with a phosphate-freedetergent by soaking in 15 % concentrated HCl for at least 10min (or with three successive rinses with dilute acid coating theentire interior surface). Following the acid treatment, rinsethree times in deionized water.
41、 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 theorganic material that might build up on its surface. Alternately,a few mL of concentrated s
42、ulfuric 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 (1) emptied; (2) rinsed with water (tapwater is permissible at this stage); (3) cleaned by
43、 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 culture glassware.6.5 AcceptabilityBefore sexual reproduction tests withseaweeds are conduct
44、ed 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 before the firsttest: (1) whether the species selected to be used reproducesacceptably; (2) wh
45、ether 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. Hazards7.1 Many materials can affect humans adversely if theprecautions taken are inadequate. Skin
46、 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, aprons, and glasses, and by using forceps to removeseaweeds from test solutions. Special prec
47、autions, 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 handling procedures (7), and chemical andphysical properties of the test materials should be stud
48、iedbefore 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 solutions, test solutions,and test organisms poses no special problems in most cases,health
49、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 because hazardous fumes might be produced.7.5 To prepare dilute acid solutions, concentrated ac
