1、Designation: D 3978 04Standard Practice forAlgal Growth Potential Testing with PseudokirchneriellasubcapitataPseudokirchneriella subcapitata12This standard is issued under the fixed designation D 3978; the number immediately following the designation indicates the year oforiginal adoption or, in the
2、 case 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.INTRODUCTIONAlgae are natural inhabitants of surface waters and are found in almost every water en
3、vironment thatis exposed to sunlight. The algae contribute to self purification (both organic and inorganic) of streamsand lakes and are necessary as food for fish and fish food organisms. When large amounts of nutrientsare available, excessive growths referred to as “blooms” can occur. Some algal b
4、looms releasesubstances toxic to fish, birds, domestic animals, and other alga. When nutrients are exhausted, thegrowth of algae and production of oxygen by photosynthesis decreases. The respiration of bacteriadecomposing the large quantity of algal cells can deplete dissolved oxygen to the extent t
5、hat fish andother oxygen consumers die. Both the abundance and composition of algae are related to water quality,with algal growth primarily influenced by the availability of nutrients.The presence of indigenous algae in a water sample suggests that they are the most fit to survivein the environment
6、 from which the sample was taken. The indigenous algae should produce biomassuntil limited from further growth by some essential nutrient. If the indigenous algal production islimited from further growth by an essential nutrient, the laboratory test alga cultured in anoncompetitive environment and r
7、esponding to the same limiting nutrient will produce parallelmaximum yield growth responses. Generally, indigenous phytoplankton bioassays are not necessaryunless there is strong evidence of the presence of long-term sublethal toxicants to which indigenouspopulations might have developed tolerance (
8、1)3.A single-indigenous algal species, dominant at the time of sampling, may not be more indicative ofnatural conditions than a laboratory species that is not indigenous to the system. The dynamics ofnatural phytoplankton blooms, in which the dominant algal species changes throughout the growthseaso
9、n, makes it quite certain that even if the indigenous algal isolate was dominant at the time ofcollection, many other species will dominate the standing crop as the season progresses.When comparing algal growth potentials from a number of widely different water sources there areadvantages in using a
10、 single species of alga. The alga to be used must be readily available and itsgrowth measured easily and accurately. It must also respond to growth substances uniformly. Becausesome algae are capable of concentrating certain nutrients in excess of their present need when they aregrown in media with
11、surplus nutrients, this factor must be taken into account in selecting the culturemedia and in determining the type and amount of algae to use. (2) showed that a blue-green algaeMicrocystis aeruginosa, cultured in a low-nitrogen concentration medium, would not grow whentransferred to medium lacking
12、nitrogen. However, when the alga was cultured in medium containingfour times as much nitrogen it was able to increase growth two-fold after transfer into nitrogen-freemedium. A green alga Pseudokirchnereilla subcapitata (formerly known as Selenastrum capricornu-tum, gave a similar response. In an an
13、alogous experiment with phosphorus, both organisms increasedfour-fold when transferred to medium lacking phosphorus. However, if algae are cultured in relativelydilute medium as recommended in the Algal Assay Procedure: Bottle Test (3) for culturingPseudokirchnereilla subcapitata, disclosed no signi
14、ficant further growth in medium lacking nitrogenor phosphorus when these were transferred from the initial medium over a wide range of inoculumsizes (4).There are several methods available for determining algal growth. Measurements of optical density,oxygen production, carbon dioxide uptake, microsc
15、opical cell counts, and gravimetric cell massdeterminations have been used, but often lack sensitivity when the number of cells is low.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Measurement of the uptake of carbon-14 in the form
16、 of bicarbonate is a sensitive method but can alsobe time-consuming. In vivo fluorescence of algal chlorophyll has been used with many types of algaeand has proved particularly useful with indigenous algae or filamentous forms not easily measured atlow concentrations by other methods. The method is
17、sensitive and measurements can be quicklyperformed. However, chlorophyll to cell mass ratio may vary significantly with growth in watersamples of different chemical composition (5). The electronic particle counter has been used forcounting and sizing nonfilamentous unialgal species (6,7). Shiroyama,
18、 Miller, and Greene (8) havedeveloped a procedure for using an electronic particle counter to count and size Anabaena flos-aquaefilaments cultured in natural waters.The need for standardization of techniques for measuring the potential for algal growth wasrecognized by the Joint Industry/Government
19、Task Force on Eutrophication (9). Thereafter, theEnvironmental Protection Agency developed, in association with industrial and university coopera-tion, a Bottle Test for assaying algal growth potential in natural water samples (3). An expanded andimproved version of the Bottle Test was published in
20、1978 (10). It is this work on which the followingtest is based.1. Scope1.1 This practice measures by Pseudokirchnereilla subcapi-tata growth response, the biological availability of nutrients, ascontrasted with chemical analysis of the components of thesample. This practice is useful for assessing t
21、he impact ofnutrients, and changes in their loading, upon freshwater algalproductivity. Other laboratory or indigenous algae can be usedwith this practice. However, Pseudokirchnereilla subcapitatamust be cultured as a reference alga along with the alternativealgal species.1.2 This standard does not
22、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. For a specificprecautionary statement, se
23、e Section 15.2. Referenced Documents2.1 ASTM Standards:4D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 3370 Practices for Sampling Water3. Summary of Practice3.1 A water sample is filtered or autoclaved and filtered,placed in a covered Erlenmeyer flask, inoculated with t
24、he testalgal species, and incubated under constant temperature andlight intensity until the increase in biomass is less than 5 % perday (generally between day 7 and 14). Nutrients may also beadded to aliquots of the sample to determine growth controllingnutrients.4. Significance and Use4.1 The signi
25、ficance of measuring algal growth potential inwater samples is that differentiation can be made between thenutrients of a sample determined by chemical analysis and thenutrients that are actually available for algal growth. Theaddition of nutrients (usually nitrogen and phosphorus singlyor in combin
26、ation) to the sample can give an indication ofwhich nutrient(s) is (are) limiting for algal growth(1,10,11,12,13,14).5. Interferences5.1 Autoclaving may cause precipitation of certain constitu-ents in the sample and elevate the pH. These precipitates arenot necessarly irreversible or unavailable as
27、nutrients. Thesample may often be clarified by equilibrating it in a CO2atmosphere followed by equilibration in air to its original pH.5.2 Toxic substances in the sample may affect the growthresponse of the algae.6. Apparatus6.1 Water Sampler, nonmetallic.6.2 Sample ContainerLinear polyethylene bott
28、les.6.3 Centrifuge.6.4 Environmental Chamber, with temperature control (246 2C) and illumination (cool white fluorescent) that provides4300 lm/m2610 %, or equivalent.6.5 Shaker, rotary, capable of 100 to 120 rpm.6.6 Flasks, Erlenmeyer, 250-mL.NOTE 1Other sizes are acceptable as long as the liquid do
29、es notexceed 50 % of the total flask volume.6.7 Flask Covers, Beakers, or Foam PlugsSome foamplugs, upon autoclaving, may release substances toxic to thetest algae. Each laboratory, when changing its source of supply,must determine whether the new closures have a significanteffect on the maximum sta
30、nding crop.1This practice is under the jurisdiction of ASTM Committee E47 on Biological Effects and Environmental Fate and is the direct responsibility of Subcommittee E47.01on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published April 2004. Originally approved in 1980
31、. Last previous edition approved 1998 as D3978-80 (Reapproved 1998).2Renamed by Gunnar Nygaard, Jirf Komrek, Jrgen Kristiansen and Olav M. Skulberg, 1986. Taxonomic designations of the bioassay alga NIVA-CHL1 (9Selenastrumcapricornutum9) and some related strains. Opera Botanica 90:5-46.3The boldface
32、 numbers in parentheses refer to the references at the end of this practice.4For 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 onth
33、e ASTM website.D39780426.8 Tubes, graduated centrifuge.6.9 Pipets, Eppendorf or equivalent, with disposable tips,0.1 or 1.0 mL.6.10 Filtration Apparatus, nonmetallic, with vacuum orpressure source.6.11 Membrane Filters, sterile 0.22-m particle size reten-tion, low-water extractable.6.12 Balance, ana
34、lytical, capable of weighing 100 g with aprecision of 60.1 mg.6.13 Autoclave.6.14 pH Meter.6.15 Light Meter, calibrated.6.16 Particle Counter and Mean Cell Volume Accessory,with 100-m aperature.7. Reagents7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indic
35、ated, it is intended thatall reagents shall conform to the specifications of the commit-tee on Analyltical Reagents of the American Chemical Soci-ety.5Other grades may be used, provided it is first ascertainedthat the reagent is of sufficiently high purity to permit its usewithout lessening the accu
36、racy of the determination.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water conformingto Specification D 1193, Type III.7.3 Calcium Chloride SolutionDissolve 1.66 g of CaCl2in 500 mL of water.7.4 Magnesium Chloride SolutionDissolve 6.08 g ofM
37、gCl26H2O in 500 mL of water.7.5 Magnesium Sulfate SolutionDissolve 3.59 g ofMgSO4in 500 mL of water.7.6 Micro Nutrient Solutions (Note 2)Dissolve the fol-lowing in 500 mL of water:NOTE 2Reagents 7.3, 7.4, 7.6, and 7.9 can be combined into one stocksolution.93 mg of boric acid (H3BO3)208 mg of mangan
38、ous chloride (MnCl24H2O)1.6 mg of zinc chloride (ZnCl2)80 mg of ferric chloride (FeCl36H2O)0.39 mg of cobalt chloride (CoCl2)3.63 mg of sodium molybdate (NaMoO42H2O)0.006 mg of cupric chloride (CuCl22H2O)150 mg of ethylenediaminetetraacetic acid(HOCOCH2)2N(CH2)2H(HOCOCH2)27.7 Potassium Phosphate Sol
39、utionDissolve 0.52 g ofK2HPO4in 500 mL of water.7.8 Sodium Bicarbonate SolutionDissolve 7.50 g ofNaHCO3in 500 mL of water.7.9 Sodium Nitrate SolutionDissolve 12.75 g of NaNO3in 500 mL of water.8. Preparation of Culture Flasks8.1 Brush the inside of flasks with a stiff bristle brush toloosen any atta
40、ched materials.8.2 Wash with nonphosphate detergent and rinse thoroughlywith tap water.8.3 Rinse with 10 % solution (9 + 1) of reagent gradehydrochloric acid (HCl) by swirling the HCl solution so thatthe entire inner surface is covered.8.4 Rinse the glassware copiously with reagent water.8.5 If an e
41、lectronic particle counter is to be used, the finalrinse should be at least 0.22-m filtered reagent water.8.6 Dry the flasks in an oven at 50C, cover, and autoclavefor 20 min at 101.325 kPa and 121C. Dry and store the cooledflasks in closed cabinets until needed.9. Culturing Techniques for Pseudokir
42、chneriellasubcapitata69.1 Prepare the culture medium as follows:9.2 Add 1 mL of each solution in 7.3-7.9 (in the order given)to approximately 900 mL of reagent water and then dilute to 1L. Adjust the pH to 7.5.9.3 If an electronic particle counter is to be used, filter themedium through a membrane f
43、ilter (0.22 m) at 50.66 kPa.9.4 Place 100-mL of sample in 250-mL Erlenmeyer flasksand close. Autoclave the prepared flasks at 121C at 101.325kPa for 20 min and allow to cool at room temperature. Store ina refrigerator until needed.9.5 Maintain the stock culture by transferring 1 mL ofa7to10-day old
44、culture to fresh medium (as described above). Thetransfer can be as often as necessary to provide an adequatesupply of algal cells at the proper growth stage for the algalgrowth potential test. Exercise extreme care to avoid contami-nation of stock cultures.9.6 To retain a unialgal culture over a lo
45、ng period of time itis advantageous to prepare medium with 1 % agar and transferalgae onto fresh plates every 4 weeks, and start fresh liquidcultures from a single colony at 4-week intervals. For regularinoculation, liquid cultures are superior since agar culturesusually are not uniform because the
46、cell layers on the agarsurface are differentially supplied with light and nutrients (as aresult of shading and diffusion).10. Sampling10.1 For maximum correlation between field and laboratoryresults, water collected for the algal growth potential testsshould be subsampled for chemical and biological
47、 study. Thesample collection method and sample size will be determinedby study objectives. Use a nonmetallic sampler. Do not reusecontainers when toxic or nutrient contamination is suspected.11. Pretreatment11.1 The method of sample pretreatment must be consideredin the interpretation of results. In
48、 cases where many microor-ganisms (protozoans, algae, bacteria, etc.) are present, a large5“Reagent Chemicals, American Chemical Society Specifications,” Am. Chemi-cal Soc., Washington, DC. For suggestions on the testing of reagents not listed bythe American Chemical Society, see “Reagent Chemicals
49、and Standards,” by JosephRosin, D. Van Nostrand Co., Inc., New York, NY, and the “United StatesPharmacopeia.”6Test algae: Pseudokirchneriella subcapitata can be obtained from the Univer-sity of Texas Culture Collection, Austin TX or the American Type CultureCollection , Rockville, MD.D3978043quantity of potential nutrients are removed by filtration. Thesemicroorganisms contain nutrients, which are not available toother algae while these organisms are living, but later becomea source of nutrients as a result of decay after death. Thus, it ispossible to measur
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