ASTM_D_3731_-_87_2012.pdf

Designation:D373187(Reapproved 2012)Standard Practices for Measurement ofChlorophyll Content of Algae in Surface Waters1This standard is issued under the fixed designation D3731;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last 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 These practices include the extraction and the measure-ment of chlorophyll a,b,and c,and pheophytin a 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 pheophytin 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 purport 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 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 submergedobjects,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 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 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 plankton 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,unless 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 routinely 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 theirpigments 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 forthis 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 D19 on Waterand are the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved Sept.1,2012.Published October 2012.Originallyapproved in 1979.Last previous edition approved in 2004 as D3731 87(2004).DOI:10.1520/D3731-87R12.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 type of sample,it is demonstrated that the chlorophyll recoveryis comparable to that obtained with tissue grinders(4).5.3 FiltersGlass-fiber filters usually provide a higher re-covery 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 degradationproducts,such as the chloro