1、Designation: D 4148 82 (Reapproved 2004)Standard Test Method forAnalysis of Phytoplankton in Surface Water by theSedgwick-Rafter Method1This standard is issued under the fixed designation D 4148; the number immediately following the designation indicates the year oforiginal adoption or, in the case
2、of revision, the 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 test method covers determining the density andtaxonomic classification of phytoplankton
3、. It is applicable bothto relatively sparse or dense phytoplankton concentrations,provided the suspended-sediment concentration is low. TheSedgwick Rafter (S-R) method requires less costly apparatusthan does the inverted microscope method but gives lessaccurate results. The inherent inaccuracy in th
4、e Sedgwick-Rafter method is due to the design of the counting chamber andcannot be circumvented by a different choice of optics. For thisreason, the S-R method is limited to the use of objective lenseshaving a working distance of approximately 1.6 mm or more.With 103 oculars the maximum overall magn
5、ification isapproximately 2503. High concentrations of suspended sedi-ment can obscure the algal cells, and thus cause interference.1.2 This test method is applicable to both freshwater andmarine samples.1.3 This standard does not purport to address all of thesafety problems, if any, associated with
6、 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. For specificprecautionary information see Section 8.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminol
7、ogy Relating to WaterD 1193 Specification for Reagent WaterD 3370 Practices for Sampling WaterD 4149 Classification for Sampling Phytoplankton in Sur-face Waters2.2 Various taxonomic keys are required for identificationof the algae. No single key is suitable for all species likely tobe encountered.
8、(See Greeson 1977; Weber 1973.)3. Summary of Test Method3.1 The microscope is calibrated to determine the field sizeon the superimposed ocular grid. A Sedgwick-Rafter chamberis filled with a preserved phytoplankton sample. After the algaesettles to the bottom, the chamber is examined microscopically
9、at 200 to 2503 for the presence of algae. Those algal cellslying within the border of the ocular grid are identified andenumerated. The tally is used to calculate the algal density incells per millilitre.4. Significance and Use4.1 Phytoplankton are basic to the food chain in all aquaticenvironments.
10、 In addition, they have long been considered tobe important indicators of water-quality conditions. Phy-toplankton data are also frequently used in the planning anddesign of water-treatment facilities and reservoirs.5. Interferences5.1 The presence of suspended sediment may obscure algalcells, makin
11、g identification difficult. Colonial forms and theoccurrence of algae in trichomes make the estimation of cellnumbers difficult. Some preservation techniques may cause aloss of flagella, hampering identification.6. Apparatus6.1 Microscope, compound, with 103 oculars and 103,253,403, and 903 objectiv
12、es, substage condenser, andmechanical stage.6.2 Ocular Micrometer, with Whipple grid.6.3 Sedgwick-Rafter Counting Cell,50by20by1mm.6.4 Stage Micrometer.6.5 Transfer Pipet, 1-mL.6.6 Microscope Slides and Cover Glasses, standard 76 by25-mm noncorrosive slides. Cover glasses, round or square,clean and
13、free of oil.7. Reagents7.1 Purity of ReagentReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Society,1This test method is under the juri
14、sdiction of ASTM Committee E47 onBiological Effects and Environmental Fate and is the direct responsibility ofSubcommittee E47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published April 2004. Originallyapproved in 1982. Last previous edition approved in 1998 as D
15、 4148 82 (1998).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.1Copyright ASTM International, 100 Barr Harbo
16、r Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.where such specifications are available.3Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.7.2 Purity of W
17、aterUnless otherwise indicated, referencesto water shall be understood to mean Type I reagent waterconforming to Specification D 1193.7.3 FormalinTo prepare the formalin preservative, mix900 mL of 37 to 40 % aqueous formaldehyde (100 % formalin)with 100 to 150 mL of 20 % surgical detergent solution
18、and 20to 30 mL of saturated cupric sulfate solution.7.4 Lugols SolutionAn alternative preservative isLugols solution. Prepare a stock Lugols solution by dissolv-ing 600 g of potassium iodide and 40 g of iodine crystals in1000 mL of water.8. Precautions8.1 Formaldehyde vapors are toxic, and the conce
19、ntratedsolution can damage exposed skin or eyes. Wear waterproofgloves and appropriate eye protection when handling concen-trated formaldehyde solutions. Work in adequately ventilatedareas.9. Sampling9.1 Collect the sample in accordance with ClassificationD 4149.9.2 Preserve the sample with either f
20、ormalin or Lugolssolution. If formalin preservative is desired, mix 40 to 50 mLof formalin preservative with each 1000 mL of sample. IfLugols solution is preferred, mix 37 mL of Lugols solutionwith each 1000 mL of sample, and store in the dark.10. Microscope Calibration10.1 Mount the ocular micromet
21、er (Whipple grid) in oneeyepiece in accordance with the manufacturers instructions forplacement.10.2 Set up the microscope and place the stage micrometeron the stage with the etched markings upper most.10.3 Focus on the ruled graduations under low power(1003). Measure and record the dimensions of th
22、e Whipplegrid to the nearest 0.01 mm. Repeat the procedure for all otherobjective/ocular combinations suitable for use with theSedgwick-Rafter cell. Often this is 2003 or 2503. At magni-fications greater than 1003, the Whipple grid should bemeasured to the nearest 0.001 mm (American Public HealthAss
23、ociation, 1976). Calculate and record the area enclosed bythe Whipple grid in square millimetres at each magnification.11. Pretreatment11.1 Some samples may require concentration or dilutionprior to analysis. The decision to concentrate or dilute issubjective and should be reached only after microsc
24、opicexamination of the sample. This can be done by preparing awet mount as follows: Mix the sample gently, then pipet a droponto a clean microscope slide, and add a cover slip. Examineat 1003 for general concentration. If desired, concentration ordilution may be performed by one of the following pro
25、cedures:11.2 Sample concentration can be accomplished in manyways; settling is the preferred way. One method is to transferthe entire sample to a graduated cylinder of sufficient capacity,noting the initial volume, and then carefully removing most ofthe supernatent by siphoning after the algae have
26、settledcompletely. Allow a time interval for settling of 4 h/cm ofdepth (Greeson, 1977). Note and record the volume of concen-trate. Another method is to weigh the water sample andcontents to the nearest 1 g, then allow the algae to settle to thebottom for 4 h/cm of depth. Note and record the initia
27、l gross ofsample weight. Then remove most of the supernatant bysiphoning. The remaining sample and container are weighedagain to determine the weight of sample discarded. Assumingan equivalence of weight and volume (1 mL = 1 g), calculateand record the volume of concentrate that will be used in late
28、ranalysis. Transfer the concentrated sample to another con-tainer, then weigh the container to determine the actualconcentration factor.11.3 Dilution of the sample may be necessary to reduce theconcentration of suspended sediment that would otherwiseobscure the algae during analysis. Occasionally a
29、sample mayhave a particularly high density of algae and require dilution.No specific guidelines are available to suggest when dilutionsshould or must be made. The analyst must decide whether ornot to dilute based on past experience. If it is decided to dilute,first mix the sample thoroughly but gent
30、ly by inverting thesample container several times. Pipet a known volume into anappropriate graduated cylinder (usually a 50 or 100-mL cylin-der is satisfactory). It is inadvisable to transfer sample volumesless than 5 mL because of the difficulty in accurately measuringvery small aliquots of sample.
31、 Dilute the sample to the desiredpoint with reagent grade water but, in any case, not beyond thecapacity of the graduated cylinder. Record the initial volume(the aliquot that was diluted) and the diluted sample volume.12. Procedure12.1 Lay the Sedgwick-Rafter cell on a flat surface with thecover gla
32、ss placed diagonally across it.12.2 Mix the sample thoroughly by turning the samplebottle end over end no less than ten times. Avoid shaking thesample as this may cause foaming or damage to delicate algae.12.3 Remove a 1-mL aliquot using a pipet and transfer it tothe Sedgwick-Rafter cell, being care
33、ful to avoid gettingbubbles under the cover glass. The cover glass must not floatabove the rim of the cell. Allow the counting cell to stand ona level surface a minimum of 24 min for the algae to settle.Often it is helpful to prepare a wet mount at the same time thatcan be used for taxonomic identif
34、ication of the algae at 4003or 9503. For example, an aliquot (50 to 100 mL) of sample canbe concentrated by centrifugation or settling, before a usefulwet mount can be made.12.4 Following settling, count the algae either by strip countor random field technique. The strip-count technique involvescoun
35、ting cells within the width of a ocular grid for the entirelength of the Sedgwick-Rafter cell. Several such “strips”3“Reagent Chemicals, American Chemical Society Specifications,” Am. Chemi-cal Soc., Washington, DC. For suggestions on the testing of reagents not listed bythe American Chemical Societ
36、y, see “Reagent Chemicals and Standards,” by JosephRosin, D. Van Nostrand Co., Inc., New York, NY, and the “United StatesPharmacopeia.”D 4148 82 (2004)2comprise the count. This technique is useful for relativelysparse samples. Another technique is to count the algal cellslying within the confines of
37、 the ocular grid in randomly spacedfields. This technique is particularly useful for dense samplesbut is cumbersome for sparse samples. There is no agreementas to the number of algae that must be counted in order toassume a statistically valid sample. Most workers suggestcounting a minimum of 100 or
38、ganisms at the very least.12.5 The counting procedures are basically the same. Twoadjacent sides of the ocular grid are designated “count” sidesand the other two “no-count” sides. Count all cells that liewithin the grid as well as those which touch a “count” side. Donot count that part of a trichome
39、 that extends outside the gridarea. When making a random field count, count a minimum of100 cells or no less than 10 fields, whichever is obtained first,but not less than 10 fields. For strip counts, count a minimumof 100 cells but make at least one complete sweep from side toside. Avoid partial swe
40、eps. Tally each taxonomic type sepa-rately.12.6 Some algae may not settle but instead rise to theunderside of the cover glass. These cells should be included inthe tally where they occur within the borders of the grid.12.7 When colonial forms are encountered, it may beimpossible to count all the cel
41、ls because of partial obscuring ofunderlying cells. In such cases, it is acceptable to calculate thetotal number of cells lying within the grid by estimation.Similarly, determine the total number of cells in filamentousforms by multiplying the mean cell length by the size oftrichome. Count the frust
42、ules containing protoplast as havingbeen living at the time of collection. Do not include emptyfrustules in the tally.13. Calculations13.1 Calculate random field count as follows:Cells/mL 5 C!N/E!P!D! (1)C 5 A/Gwhere:C = Calibration factor,A = area of S-R cell, mm2, andG = field (grid) area, mm2.P 5
43、 S/V (2)where:P = preservative factor,S = sample plus preservative volume, mL, andV = sample volume, mL.D 5 M/O (3)where:D = concentration or dilutions factor,M = concentrated or diluted volume, mL, andO = original sample volume, mL.13.2 Calculate strip counts as follows:Cells/mL 5 C!N/E!P!D! (4)C 5
44、 A/Bwhere:B = area of strip, mm2.P 5 S/V (5)D 5 M/Twhere:T = sample volume prior to concentration per dilution.14. Report14.1 Results shall be reported as the number of cells permillilitre for each taxonomic type.15. Precision and Bias15.1 Because of the taxonomic complexity of the samples,the true
45、value, and therefore the accuracy, cannot be deter-mined.15.1.1 The precision of the count is related to the number oforganisms counted, and is determined by calculating the squareroot of the tally. For example: if 100 cells (or units) areencountered in ten fields, the precision of the count would b
46、e100 6=100 = 100 6 10, or 100 6 10 %. The final value, interms of cells (or units) per millilitre can be determined by theappropriate conversion factor. In the example, the precision ofthe final value would be 610 %.ASTM International takes no position respecting the validity of any patent rights as
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49、eive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serv
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