1、Designation: D3978 04 (Reapproved 2012)Standard Practice forAlgal Growth Potential Testing with Pseudokirchneriellasubcapitata1,2This standard is issued under the fixed designation D3978; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revis
2、ion, 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.INTRODUCTIONAlgae are natural inhabitants of surface waters and are found in almost every water environmentthat i
3、s exposed to sunlight. The algae contribute to self purification (both organic and inorganic) ofstreams and lakes and are necessary as food for fish and fish food organisms. When large amounts ofnutrients are available, excessive growths referred to as “blooms” can occur. Some algal bloomsrelease su
4、bstances toxic to fish, birds, domestic animals, and other alga. When nutrients are exhausted,the growth 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 that fish andoth
5、er 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 from which the
6、 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 responding to th
7、e 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 (1)3.Asingle-ind
8、igenous 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 growthseason, makes it quit
9、e 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 single species
10、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 surplus nutrient
11、s, 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 nitrogen. Howeve
12、r, 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 Selenastrumcapricornutum, gave a similar response. In an analogous experiment
13、 with phosphorus, both organismsincreased four-fold when transferred to medium lacking phosphorus. However, if algae are culturedin relatively dilute medium as recommended in the Algal Assay Procedure: Bottle Test (3) forculturing Pseudokirchnereilla subcapitata, disclosed no significant further gro
14、wth in medium lackingnitrogen or phosphorus when these were transferred from the initial medium over a wide range ofinoculum sizes (4).There are several methods available for determining algal growth. Measurements of optical density,oxygen production, carbon dioxide uptake, microscopical cell counts
15、, and gravimetric cell massdeterminations have been used, but often lack sensitivity when the number of cells is low.Measurement of the uptake of carbon-14 in the form of bicarbonate is a sensitive method but can alsoCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken
16、, PA 19428-2959. United States1be 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 sensitive and measu
17、rements 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, Miller, and Greene
18、 (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 Task Force on Eutro
19、phication (9). Thereafter, theEnvironmental Protection Agency developed, in association with industrial and universitycooperation, a Bottle Test for assaying algal growth potential in natural water samples (3).Anexpanded and improved version of the Bottle Test was published in 1978 (10). It is this
20、work on whichthe following test 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 the impact ofnutrients,
21、 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 purport to address all
22、 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, see Section 15.2. Refere
23、nced Documents2.1 ASTM Standards:4D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD3370 Practices for Sampling Water from Closed Conduits3. Summary of Practice3.1 A water sample is filtered or autoclaved and filtered,placed in a covered Erlenmeyer flask, inoculated with the t
24、estalgal 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 significa
25、nce 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 combinatio
26、n) 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 nut
27、rients. 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 bottles
28、.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 does
29、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,1This practice is under the jurisdiction of ASTM Committee E47 on Biological Effects a
30、nd Environmental Fate and is the direct responsibility of Subcommittee E47.01on Aquatic Assessment and Toxicology.Current edition approved Sept. 1, 2012. Published October 2012. Originally approved in 1980. Last previous edition approved 2004 as D3978-04 (Reapproved 1998). DOI:10.1520/D3978-04R12.2R
31、enamed 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 numbers in parentheses refer to the references at the end of this pra
32、ctice.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 onthe ASTM website.D3978 04 (2012)2must determine whether the new closures
33、 have a significanteffect on the maximum standing crop.6.8 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 sizeretention, low-water
34、 extractable.6.12 Balance, analytical, 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
35、 tests. Unless otherwise indicated, 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
36、 usewithout lessening the accuracy of the determination.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water conformingto Specification D1193, Type III.7.3 Calcium Chloride SolutionDissolve 1.66 g of CaCl2in500 mL of water.7.4 Magnesium Chloride
37、 SolutionDissolve 6.08 g ofMgCl26H2O 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 follow-ing 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
38、acid (H3BO3)208 mg of manganous 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)
39、27.7 Potassium Phosphate SolutionDissolve 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 NaNO3in500 mL of water.8. Preparation of Culture Flasks8.1 Brush the inside of flasks with a stiff bris
40、tle brush toloosen any attached 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 wit
41、h reagent water.8.5 If an electronic 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. Culturi
42、ng Techniques for Pseudokirchneriellasubcapitata9.1 Prepare the culture medium as follows:9.2 Add 1 mLof 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 theme
43、dium through a membrane filter (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 transferri
44、ng 1 mL ofa7to10-day old 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 u
45、nialgal culture over a long 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
46、 not uniform because the 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 fo
47、r chemical and biological 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.5“Reagent Chemicals,American Chemical Society Specifications,”Am. Chemi-cal Soc., Wa
48、shington, DC. For suggestions on the testing of reagents not listed bytheAmerican Chemical Society, see “Reagent Chemicals and Standards,” by JosephRosin, D. Van Nostrand Co., Inc., New York, NY, and the “United StatesPharmacopeia.”D3978 04 (2012)311. Pretreatment11.1 The method of sample pretreatme
49、nt must be consideredin the interpretation of results. In cases where many microor-ganisms (protozoans, algae, bacteria, etc.) are present, a largequantity 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 measure a high concentration of algae during a“bloom” but observe a low dissolved algal growth potential.Nutrients can also be derived from nonorgani
