1、Designation: E 1366 02 (Reapproved 2007)Standard Practice forStandardized Aquatic Microcosms: Fresh Water1This standard is issued under the fixed designation E 1366; 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 (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers procedures for obtaining dataconcerning toxicity and other effects of a test material to amulti-troph
3、ic level freshwater community.1.2 These procedures also might be useful for studying thefate of test materials and transformation products, althoughmodifications and additional analytical procedures might benecessary.1.3 Modification of these procedures might be justified byspecial needs or circumst
4、ances. Although using appropriateprocedures is more important than following prescribed pro-cedures, results of tests conducted using unusual proceduresare not likely to be comparable to results of many other tests.Comparison of results obtained using modified and unmodifiedversions of these procedu
5、res might provide useful informationconcerning new concepts and procedures for conducting multi-trophic level tests.1.4 This practice is arranged as follows:SectionReferenced Documents 2Terminology 3Summary of Practice 4Significance and Use 5Apparatus 6Facilities 6.1Container 6.2Equipment 6.3Hazards
6、 7Microcosm Components 8Medium 8.1Medium Preparation 8.2Sediment 8.3Microcosm Assembly 8.4Test Material 9General 9.1Stock Solution 9.2Nutrient Control 9.3Test Concentration(s) 9.4Test Organisms 10Algae 10.1Animals 10.2Specificity of Organisms 10.3Sources 10.4Algal Culture Maintenance 10.5Animal Cult
7、ure Maintenance 10.6SectionProcedure 11Experimental Design 11.1ProcedureInoculation 11.2Culling 11.3Addition of Test Material 11.4Measurements 11.5Reinoculations 11.6Analytical Methodology 12Data Processing 13Calculations of Variables from Measurements 14Statistical Analyses 15Acceptability of Test
8、16Interpretation of Results 17Report 18Annex Annex A1AppendicesRelationship of Media Appendix X1Data Sheets Appendix X2Statistical Guidance Appendix X31.5 The values stated in SI units are to be regarded as thestandard. The values given in parentheses are for informationonly.1.6 This standard does n
9、ot 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. Specific hazardstatements are given in
10、 Section 7.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterD 3978 Practice for Algal Growth Potential Testing withPseudokirchneriella subcapitataE 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE 943 Ter
11、minology Relating to Biological Effects and En-vironmental FateE 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their Uses1This practice is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of Subcommi
12、tteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved Oct. 1, 2007. Published October 2007. Originallyapproved in 1990. Last previous edition approved in 2002 as E 1366 02.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serv
13、iceastm.org. For Annual Book of ASTMStandards volume 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.E 1192 Guide for Conducting Acute Toxicity Tests onAque
14、ous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE 1193 Guide for Conducting Daphnia magna Life-CycleToxicity TestsIEEE/SI 10 American National Standard for Use of theInternational System of Units (SI): The Modern MetricSystem3. Terminology3.1 The words “must,” “should
15、,”“ may,” “can,” and “might”have very specific meanings in this practice. “Must” is used toexpress an absolute requirement, that is, to state that the testought to be designed to satisfy the specific condition, unless thepurpose of the test requires a different design. “Must” is onlyused in connecti
16、on with factors that directly relate to theacceptability of the test (see Section 17). “Should” is used tostate that the specified condition is recommended and ought tobe met in most tests. Although a violation of one “should” israrely a serious matter, violation of several will often render theresu
17、lts questionable. Terms such as “is desirable,” “is oftendesirable,” and “might be desirable” are used in connectionwith less important factors. “May” is used to mean “is (are)allowed to,”“ can” is used to mean “is (are) able to,” and“might” is used to mean “could possibly.” Thus, the classicdistinc
18、tion between “may” and “can” is preserved, and “might”is never used as a synonym for either “may” or “can.”3.2 For definitions of other terms used in this practice, referto Guide E 729, Terminology E 943, and Guide E 1023. For anexplanation of units and symbols, refer to IEEE/SI 10.3.3 Definitions o
19、f Terms Specific to This Standard:3.3.1 algal biovolume, nan estimate of the total volume ofalgal cells (31043/mL) (see 14.1.10).3.3.2 available algae, nan estimate of the volume of algae(31043/mL) presumed available to the Daphnia (see14.1.10).3.3.2.1 DiscussionThe estimate is calculated from thenu
20、merical abundance of each species of algae, its nominalvolume, and an availability factor based on its size and growthcharacteristics (see 14.1.10). Small algal cells are presumed100 % available and large, filamentous forms are presumed 1to 20 % available. Species that attach to sediment or walls ar
21、epresumed to be less available than planktonic forms.3.3.3 axenic, adja culture of organisms growing withoutneighbors, that is, pure culture free from contaminant organ-isms (see gnotobiotic (12)3).3.3.4 community metabolism, nthe oxygen or carbonbalance of the entire community.3.3.4.1 DiscussionIn
22、this microcosm, community me-tabolism is estimated by the gain in oxygen during the lightedperiod (an estimate of net photosynthesisP) and the loss ofoxygen during the dark period (an estimate of respirationR).When expressed as a P/R ratio, a value of 1 indicates thatautotrophic processes are domina
23、nt; a value of 1.8 mm), three large with eggs, three without eggs, and tensmall (0.7 mm); 12 amphipods (six adults and six small), andsix ostracods.FIG. 4 Semicontinuous Algal Culture Unit (10.5.4.1)E 1366 02 (2007)1211.2.4 Stock CulturesStock cultures should be maintainedfor reinoculation during th
24、e test (11.6).11.3 Culling and AssignmentOn Day 7, the necessarynumber of microcosms should be selected for the test. Anymicrocosms in cracked jars should be eliminated becausecracks tend to grow and leak. Data through Day 7 on thefollowing measurements (see 12.5) should be used to deter-mine culls:
25、 change in dissolved oxygen from a.m. to p.m., pH,number of Daphnia, the number of Selenastrum and Chlamy-domonas, and the dominant algae early in microcosm devel-opment. The necessary number of test systems with the leastvariation from one another in these measurements should besaved and the others
26、 discarded or used for other purposes (forexample, as amphipod and ostracod cultures).11.3.1 Treatment AssignmentThe selected microcosmsshould be numbered and a random number table used to assigneach microcosm to a treatment and to a position separately onthe light table (Fig. 1). For table position
27、 the microcosms areassigned to 6 blocks, and randomly assigned to each. Treat-ments must be randomly assigned to individual test chamberlocations. A randomized block design (with each treatmentbeing present in each block, which may be a row or arectangle) is preferable to a completely randomized des
28、ign.Microcosms are then numbered consecutively as in Fig. 1.NOTE 3Microcosms that crack while the experiment is in progressmay be transferred to new sterile containers after a thorough scraping.Occasionally this give rise to outliers; examination of the data determinestheir eventual inclusion or exc
29、lusion.11.4 Addition of Test Material:11.4.1 Single AdditionTest material should be added onDay 7 after treatment assignment. Each microcosm should bestirred with a glass rod after test chemical or solvent, or both,are added.11.4.2 Multiple AdditionThe test material may be addedbiweekly or weekly af
30、ter sampling. The volume of eachmicrocosm should be measured again before test materialaddition and the addition calculated to provide the appropriateconcentration. If residual test material in the microcosm is tobe measured, the samples should be removed prior to the newaddition. It is recommended
31、that medium T82MV not be addedto compensate for sample removal, any medium additionwould serve as a source of nutrients and eliminate the need fornutrient recycling to occur for continued photosynthesis. How-ever, medium addition may be necessary if large samples mustbe removed for test chemical ana
32、lyses (See 11.5.6.2(e) Dis-tilled water is not added because it could cause osmoticimbalances.11.5 Measurements:NOTE 4Each task should be assigned to a person (see Appendix Fig.X2.4). All data should be written on computer data sheets as they arecollected. Data sheet formats are suggested inAppendix
33、, Figs. X2.1-X2.9.Microcosms should be grouped by treatments on the data sheets. When amicrocosm breaks or data are missing for a measurement, an entry shouldstill be made that includes experiment designation, subcode, microcosmnumber and test day number, and the data portion left blank.NOTE 5Separa
34、te probes, and other equipment (containers, scrapers,stirrers, and syringes) should be used for each treatment. It is recom-mended that where possible all sampling devices be autoclaved orotherwise sterilized prior to the beginning of each test. Although aseptictechniques are not required for most o
35、f the sampling procedures, careshould be exercised to avoid contamination with other organisms; forexample, none of the sampling devices should be used with lake orsediment samples. Care should be taken so that mixing of even smallamounts of test and control microcosms is avoided.11.5.1 Sampling Fre
36、quencySampling and measurementof organism abundances and physical factors should be donetwice each week until the end of the test, usually Day 63.Measurements should be done on the same test day for eachtest if it is desirable to compare data between tests. Tuesdaysand Fridays are the most convenien
37、t days because the first twoTABLE 6 Medium T85MVK CompositionDifferences fromT82MVACompoundMolecularWeightConcentrationmM Element mg/LNaNO385.0 0.05 Nitrogen 0.70KH2PO4136.0 0.004 Phosphorus 0.123Added (Keatings Metals) M g/LNaBr 102.89 0.626 Bromine 50SrCl26H2O 266.52 1.141 Strontium 100RbCl 120.92
38、 1.170 Rubidum 100LiCl 42.39 14.41 Lithium 100KI 166.00 0.0392 Iodine 5.0SeO2110.96 0.0127 Selenium 1.0NH4VO3116.93 0.00983 Vanadium 0.5ABasal medium is prepared as for T82MV (Table 1, Section 8) except that110the volume of Solution A (nitrate) is added.TABLE 7 T85MVKDaphnia Culturing MediumMasterSo
39、lutionSalt mL/L mL/18 L Concentration/mM(Final Solution)A NaNO30.5 9.0 0.05B MgSO47H2O 1.0 18.0 0.1D CaCl2H2O 10.0 180.0 1.0E NaCl 15.0 270.0 1.5HAl2(SO4)318H2O 1.0 18.0 0.0048INa2SiO39H2O 5.0 90.0 0.080CA0.04 0.72 .KA0.05 0.9 .VitaminsA1.0 18.0 .Si-10XA5.0 90.0 .KeatingsA1.0 18.0 .HClAto pH 7 to pH
40、 7 .AThe specified amounts of these solutions should be added after the major saltsolution is autoclaved and cooled. This delays precipitation prior to dispensing.TABLE 8 Wheat Grass Medium10for Culture of Protozoa andRotifersI. Inorganic Stock SolutionsSolutionNumberSolution Stock ConcentrationmL/L
41、 CultureMedium1Na2HPO470.9825gL1(0.5M) 10 mLor orNa2HPO47H2O 134.04 g/L (0.5 M) 10 mL2KH2PO468.04 g/L (0.5 M) 10 mL3 NaOH 4.0 g/L (0.1 M) 20 mL4 KCl 7.48 g/L 5 mLNaCl 23.40 .MgSO47H2O 26.64 .CaCl22H2O 2.78 .MnCl24H2O 0.40 .NH4Cl 38.22 .II. Dried Grass or Wheat Grass Powder102.5 g/LE 1366 02 (2007)13
42、oxygen measurements (of the 3-point method) can be takenMonday and Thursday; thus, no weekend work is required onthis schedule.11.5.2 TurbidityPrior to dissolved oxygen determinations,approximately 5 mL should be withdrawn from each unstirredmicrocosm for measurement of turbidity. If the instrument
43、doesnot provide a digital output of absorbance, t, the percenttransmittance should be recorded, and converted to opticaldensity during the computerized data processing (14.1.7).11.5.3 VolumeAn external calibrated rod may be used toestimate volume. A30-cm rod may be calibrated to a samplemicrocosm ja
44、r containing 200 g of microcosm sediment sandand 500 mL of medium or water. The first gradation is markednext to the liquid level and designated 500 mL.Aliquots of 100mL of water are added and the stick marked each time until3500 mL is reached. On sampling days, volume can bemeasured by holding the
45、calibrated rod against a microcosmand noting on a data sheet which gradation is closest to themicrocosm water level.11.5.4 Dissolved OxygenDissolved oxygen concentra-tions should be measured 3 times: before lights-on (8:00 to9:00 a.m.) and late afternoon (4:00 to 5:30 p.m.) before asampling day, and
46、 the predawn before sampling (8:00 to 9:00a.m.). Separate probes, marked with tape or symbol should beused for each test material. They are calibrated and stored inthe mouth of reagent bottles, unplugged from the meter. Foruse, probes should be plugged into the meter, standardized andreadings taken.
47、 If the test involves a control group and morethan one concentration of a test material, all replicates with testmaterial are read using the same probe, the group with thelowest concentration of test material being measured first. Theprobe is rinsed with distilled water between groups. Controlreplic
48、ates are always measured only with the control probe.The a.m. measurements are taken before the lights are on usinga flashlight to observe meter readings and microcosms. Thep.m. measurements are taken late in the afternoon aftermaximum photosynthetic activity has been achieved. Dis-solved oxygen is
49、measured while moving the probe in acircular motion. A self-stirring probe may be used.11.5.5 pHpH values should be measured before lights-oneach sampling day. Separate probes are used for the control andtreatments, as specified for dissolved oxygen probes.11.5.6 Sampling.11.5.6.1 Method of MixingThe sides and bottom of themicrocosm jars should be scraped as completely as possibleand stirred vigorously with a prerinsed and autoclaved rubberscrapper (rubber policeman, see Fig. 5) attached to a glass rod.11.5.6.2 Sampling and Organism Enumeration
