1、Designation: D 3731 87 (Reapproved 2004)Standard Practices for Measurement ofChlorophyll Content of Algae in Surface Waters1This standard is issued under the fixed designation D 3731; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision,
2、 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 These practices include the extraction and the measure-ment of chlorophyll a, b, and c, and pheophytin a
3、 in freshwaterand marine plankton and periphyton. Three practices areprovided as follows:1.1.1 Spectrophotometric, trichromatic practice for measur-ing chlorophyll a, b, and c.1.1.2 Spectrophotometric, monochromatic practice for mea-suring chlorophyll a corrected for pheophytin a; and formeasuring p
4、heophytin a.1.1.3 Fluorometric practice for measuring chlorophyll acorrected for pheophytin a; and for measuring pheophytin a.1.2 The values stated in SI units are to be regarded as thestandard. The values given in parentheses are provided forinformation purposes only.1.3 This standard does not purp
5、ort to address all of thesafety problems, 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 precau-tionary statements are given
6、in Section 7.2. Terminology2.1 Definitions:2.1.1 planktonnonmotile or weakly swimming organisms,usually microscopic, that drift or are carried along by currentsin surface waters, commonly consisting of bacteria, algae,protozoa, rotifers, and microcrustacea.2.1.2 periphytonmicroorganisms growing on s
7、ubmergedobjects, commonly consisting of bacteria, algae, protozoa, androtifers.3. Summary of Practices3.1 The chlorophyll and related compounds are extractedfrom the algae with 90 % aqueous acetone. The concentrationof the pigments is determined by measuring the light absorp-tion or fluorescence of
8、the extract.4. Significance and Use4.1 Data on the chlorophyll content of the algae have thefollowing applications:4.1.1 To provide estimates of algal biomass and productiv-ity.4.1.2 To provide general information on the taxonomiccomposition (major groups) of the algae, based on the relativeamounts
9、of chlorophyll a, b, and c, and the physiologicalcondition of algal communities, which is related to the relativeabundance of pheopigments.4.1.3 To determine long-term trends in water quality.4.1.4 To determine the trophic status of surface waters.4.1.5 To detect adverse effects of pollutants on pla
10、nkton andperiphyton in receiving waters.4.1.6 To determine maximum growth rates and yields inalgal growth potential tests.5. Interferences and Special Considerations5.1 Pigment ExtractionThe chlorophylls are only poorlyextracted, if at all, from some forms of algae, such as thecoccoid green algae, u
11、nless the cells are disrupted, whereasother algae, such as the diatoms, give up their pigments veryreadily when merely steeped in acetone. Since natural commu-nities of algae usually consist of a wide variety of taxa thatdiffer in their resistance to extraction, it is necessary to disruptthe cells r
12、outinely to ensure maximum recovery of the chloro-phylls. Failure to do so may result in a systematic underesti-mation of 10 % or more in the chlorophyll content of thesamples. (1, 2, 3)25.2 GrindersThe cells of many common coccoid greenalgae resist disruption by most methods, but usually yield thei
13、rpigments after maceration with a tissue grinder. The routineuse of grinders, therefore, is recommended. Glass-to-glassgrinders are more rigorous in disrupting cells in planktonconcentrated by centrifugation, and in periphyton scrapings,than are TFE-fluorocarbon-to-glass grinders, and their use fort
14、his purpose is preferred. However, TFE fluorocarbon-to-glassgrinders perform well with glass-fiber filters. Other cell dis-ruption methods, such as sonication, may be used if, for each1These practices are under the jurisdiction of ASTM Committee E47 onBiological Effects and Environmental Fate and ar
15、e the direct responsibility ofSubcommittee E47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published April 2004. Originallyapproved in 1979. Last previous edition approved in 1998 as D 3731 - 79 (1998).2The boldface numbers in parentheses refer to the list of refe
16、rences at the end ofthis standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.type of sample, it is demonstrated that the chlorophyll recoveryis comparable to that obtained with tissue grinders (4).5.3 FiltersGlass-fiber filters
17、usually provide a higherrecovery of chlorophyll than is obtained with membrane filterswhen extraction-resistant algae are present in the samples, andshould be employed routinely (4).5.4 Chlorophyll-Related PigmentsNaturally occurring,structurally related chlorophyll precursors and degradationproduct
18、s, such as the chlorophyllides, pheophytins, andpheophorbides, commonly occur in pigment extracts and mayabsorb light in the same region of the spectrum as thechlorophylls. These compounds may interfere with the analysisby indicating falsely high chlorophyll concentrations.5.4.1 This practice includ
19、es a correction for pheophytin aonly. Pheophytin a is similar in structure to chlorophyll a, butlacks the magnesium atom (Mg) in the porphyrin ring. Themagnesium can be removed from chlorophyll in the laboratoryby acidifying the extract. When a solution of pure chlorophylla is converted to pheophyti
20、n a by acidification, the absorptionpeak is reduced to approximately 60 % of its original value andshifts from 664 to 665 nm, resulting in a before:after acidifi-cation absorption peak ratio (OD664/OD665) of 1.70. Thisphenomenon is utilized in correcting the apparent concentra-tion of chlorophyll a
21、for the presence of pheophytin a.Unwanted degradation of chlorophyll to pheophytin in thephytoplankton on filters, or in periphyton samples, or in theacetone extract, by the occurrence of acidic conditions can beprevented by the addition of a magnesium carbonate suspen-sion to the plankton sample be
22、fore filtering or to the periphytonsamples before grinding, and by adding a small amount of asodium bicarbonate solution to the aqueous acetone when it isprepared. Addition of magnesium carbonate may also aid inclarifying the samples following steeping (5).5.5 TurbidityThe optical density of the ext
23、ract is mea-sured at 750 nm to correct for turbidity.5.6 Spectrophotometer ResolutionThe absorption peak ofacetone solutions of chlorophyll extracts is relatively narrow,and a spectrophotometer with a resolution of 2 nm or better isrequired to obtain accurate results. If instruments of lowerresoluti
24、on are employed, the concentration of chlorophyll amay be significantly underestimated depending on the bandwidth. At a spectral band width of 20 nm, the error in theestimate of the chlorophyll a concentration may be as large as40 %.5.7 Fluorometer FiltersIn the fluorometric practice, inter-ferences
25、 from light emitted by chlorophyll b and chlorophyll care greatly reduced by the use of a sharp cut-off red filter3thatblocks all light with a wavelength of less than 650 nm (6).5.8 Light Sensitivity of ExtractsChlorophyll solutions de-grade rapidly in strong light. Work with these solutions,therefo
26、re, should be carried out in subdued light, and allvessels, tubes, and so forth, containing the pigment extractsshould be covered with aluminum foil or other opaque sub-stance.6. Apparatus6.1 Filters, Glass-fiber filters, providing quantitative reten-tion of particles equal to or greater than 0.45 m
27、 in diameter.6.2 Filtering Apparatus suitable for use with glass-fiberfilters.6.3 Tissue HomogenizerTissue grinder consisting of amotor-driven pestle and enclosing glass tube (glass to glass orTFE-fluorocarbon-to-glass grinder).46.4 Spectrophotometer suitable for use over the range from600 to 750 nm
28、, with a resolution of 2 nm or better, andequipped with sample cells having a light path of 1, 5, and 10cm, with a capacity of 10 mL or less.6.5 Fluorometer (Optional):6.5.1 Spectrophotofluorometer that provides an excitationwavelength of 430 nm and detection of emission over the rangefrom 600 to 70
29、0 nm, or:6.5.2 Filter Fluorometer equipped with a blue light sourceand blue excitation filter5and a sharp cut off filter36.6 Centrifuge that can provide a centrifugal force of 1000g; head with swing-out buckets preferred.6.7 Centrifuge Tubes, screw-cap or stoppered, conical,graduated, 15-mL. Avoid c
30、ap liners soluble in acetone andneoprene rubber stoppers.7. Reagents and Materials7.1 Aqueous Acetone, 90 %Add 1 volume of distilledwater to 9 volumes of reagent grade acetone. Add 5 drops of 1N sodium bicarbonate solution per litre. (Cautionthe vol-ume:volume relationship between the acetone and wa
31、ter mustbe strictly followed to prevent shifts in the absorption peaks.)7.2 Hydrochloric Acid (1 N)Add one volume of concen-trated hydrochloric acid (HCl, sp gr 1.19) to eleven volumes ofdistilled water.7.3 Magnesium Carbonate SuspensionAdd1goffinelypowdered magnesium carbonate to 100 mL of distille
32、d water ina stoppered Erlenmeyer flask. Shake immediately before use.7.4 Sodium Bicarbonate Solution (1 N)Prepare by dis-solving 8.4 g of sodium bicarbonate in 100 mL of distilledwater.8. Sampling8.1 Plankton:8.1.1 CollectionCollect samples with a water bottle, dia-phragm pump, or other suitable dev
33、ice. To protect the chloro-phyll from degradation prior to extraction and analysis, imme-diately add 1 mL of magnesium carbonate suspension per L ofsample, and protect from the direct sunlight.8.1.2 ConcentrationImmediately concentrate the plank-ton by filtering or by centrifuging for 20 min at 1000
34、 g. Toavoid cell damage and loss of contents during filtration, do notexceed a vacuum of12 atm (50 kPa). After centrifuging, checkthe samples for buoyant cells that may resist sedimentation.3Corning CS-2-64 filter or its equivalent, has been found suitable for thispurpose. Available from Corning Gla
35、ss Works, 388 Beartown Rd., Painted Post, NY14870.4Kontes type C, glass-to-glass grinder or its equivalent, has been found suitablefor this purpose. Available from Kontes Manufacturing Co., Spruce St., Vineland,NJ 08360.5Corning CS-5-60 filter has been found satisfactory. Equivalent filters may beus
36、ed.D 3731 87 (2004)28.1.3 Holding TimeSamples that cannot be concentratedimmediately after collection may be held at 0 to 4C in the darkfor 24 h before the plankton are concentrated. The centrifugateor residue on the filter may be stored in the dark at 20C for30 days before extracting the pigment.8.
37、2 Periphyton:8.2.1 CollectionTake samples from natural or artificialsubstrates and immediately ice, freeze, or place in cold (iced)aqueous acetone in the dark.8.2.2 Holding TimeIced samples may be held 24 h beforethey are further processed. Frozen samples may be heldat 20C for 30 days.9. Pigment Ext
38、raction9.1 Algal cells in plankton concentrates (from centrifuga-tion or filtration) and periphyton scrapings are disrupted bygrinding in a tissue homogenizer for 3 min at approximately500 r/min in 4 to 5 mL of 90 % aqueous acetone. Use aglass-to-glass tissue grinder for macerating plankton concen-t
39、rate obtained by centrifugation and for macerating periphytonscrapings. A TFE-fluorocarbon-to-glass or glass-to-glassgrinder may be used to macerate plankton concentrated onglass-fiber filters.9.2 Wash the homogenate into a vial or into a 15-mLcentrifuge tube, rinse the pestle and grinding tube with
40、 a smallamount of aqueous acetone, bring the volume of the extract to10 mL by adding 90 % aqueous acetone, cap or stopper thetube, and mix and place the material in the dark at 4C to steep.9.3 Steep not less than 15 min or more than 24 h. Mix thehomogenate by inverting the tube several times, and cl
41、arify theextract by centrifuging 20 min at 1000 g, or by filtering. If theclarified extract is not analyzed immediately, store in the darkat 20C in a tightly stoppered tube.9.4 After clarification, decant the extract directly into acuvette or a screw-cap or stoppered tube. If the analysis can notbe
42、carried out immediately, the extract can be stored for 1 yearwithout appreciable chlorophyll degradation if held in the darkat 20C.10. Extract Analysis10.1 The two practices of extract analysis commonly em-ployed are visible spectrophotometry and fluorometry (6, 7).Each practice has its advantages a
43、nd disadvantages. Thetrichromatic, spectrophotometric practice, has the advantage ofproviding a simple procedure for the simultaneous estimationof chlorophyll a, b, and c, which, at the current state oftechnology, cannot be obtained as easily by the fluorometricpractice, but does not correct for chl
44、orophyll degradationproducts. The monochromatic, spectrophotometric practicecorrects the chlorophyll a for pheophytin, a, but does notmeasure chlorophyll b and c. The fluorometric practice, is oneto three orders of magnitude more sensitive than the spectro-photometric practices, when using the instr
45、umentation com-monly employed for chlorophyll analyses, but the practices forsimultaneously measuring chlorophyll b and c are much morecomplex.10.2 Spectrophotometric, Trichromatic Practice:10.2.1 The chlorophyll concentration is a function of theabsolute optical density (OD) of the extract at the s
46、pecifiedwavelengths, rather than the relative OD as is commonly thecase in colorimetric analyses. For quantitative chlorophylldeterminations, it is essential, therefore, to check the instru-ment or chart OD readings, or both, at several wavelengths inthe range from 600 to 750 nm across the full span
47、 of theabsorbance (OD = 0.01.0), using a set of filters6of knownoptical density.10.2.2 Transfer the extract to a sample cell and measure theoptical density at 750, 664, 647, and 630 nm. If possible,choose a cell-path length or dilution to provide an OD664greater than 0.2 and less than 1.0.10.2.3 Sub
48、tract the OD750 from each of the other ODs.Then divide by the cell-path length in centimetres.10.2.4 Calculate the concentration of chlorophyll a, b, and cin the extract by inserting the 1-cm OD664, OD647, andOD630 into the following Jeffrey and Humphrey equations (6):Chl a, mg/L 5 11.85OD664! 2 1.5
49、4OD647! 2 0.08OD630!(1)Chl b, mg/L 5 21.03OD647! 2 5.43OD664! 2 2.66OD630!(2)Chl c, mg/L 5 24.52OD630! 2 1.67OD664! 2 7.60OD647!(3)10.2.5 Express the concentration of pigments in a planktonsample as milligrams per cubic metre (mg/m3) and calculate asfollows:Chl a, mg/m35Ca 3 EG(4)where:Ca = concentration of chlorophyll a in the extract, mg/L,E = extract volumes, L, andG = grab sample volume, m3.10.2.6 Express the concentration of pigments in a periphy-ton sample as milligrams per square metre (mg/m2) andcalculate as follows:Chl a, mg/m25Ca 3 EA(
