1、Designation: E645 13Standard Practice forEvaluation of Microbicides Used in Cooling Water Systems1This standard is issued under the fixed designation E645; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、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 This practice outlines a procedure for evaluating theefficacy of microbicides (algicides, bactericides, and fungi-cides) that will be
3、 used for controlling microbial growth incooling water systems. The microbicides will be evaluatedusing simulated or real cooling tower water against (1)microbes from cooling water, (2) microbes in microbiologicaldeposits (biofilms) from operating cooling systems, or (3)microorganisms known to conta
4、minate cooling water systems,or a combination thereof. This practice should be performed byindividuals familiar with microbiological techniques.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purpo
5、rt to address all of thesafety concerns, 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.2. Referenced Documents2.1 ASTM Standards:2D373
6、1 Practices for Measurement of Chlorophyll Content ofAlgae in Surface Waters (Withdrawn 0)3D4012 Test Method forAdenosine Triphosphate (ATP) Con-tent of Microorganisms in WaterD4412 Test Methods for Sulfate-Reducing Bacteria in Waterand Water-Formed DepositsE1054 Test Methods for Evaluation of Inact
7、ivators of Anti-microbial AgentsE1326 Guide for Evaluating Nonconventional Microbiologi-cal Tests Used for Enumerating BacteriaE1427 Guide for Selecting Test Methods to Determine theEffectiveness of Antimicrobial Agents and Other Chemi-cals for the Prevention, Inactivation and Removal ofBiofilm (Wit
8、hdrawn 2009)3E2756 Terminology Relating to Antimicrobial and AntiviralAgents3. Terminology3.1 For definitions of terms used in this practice, seeTerminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 algicide, na chemical agent that kills algae; unicel-lular or filamentous chloro
9、phyll-containing plants.3.2.2 bactericide, na physical or chemical agent that killsbacteria, but not necessarily bacterial spores.3.2.3 biofilm, na dynamic, self-organized accumulation ofmicroorganisms and environmental by-products immobilizedon a substrate and embedded in an organic polymer matrix.
10、3.2.4 cooling system, nequipment and coolant used for theremoval of heat from processes, equipment, or both.3.2.4.1 DiscussionThe most common medium used forremoval or transfer of heat is water. The heated water then canbe discharged into a receiving body (once through coolingsystem) or it can be co
11、oled and reused (recirculating coolingsystem).3.2.5 cooling tower, na structure used to dissipate heat inopen recirculating cooling systems.3.2.6 cooling water, nany water-based solution that ab-sorbs and transfers heat in a heat exchange system.3.2.7 fungicides, na physical or chemical agent that k
12、illsfungi; that is, vegetative mycelia and/or budding yeasts includ-ing spores and/or conidia.3.2.8 microbial biofouling, nthe unwanted accumulationof bacterial, fungal, or algal cells, or any combination thereofand their products on surfaces.3.2.8.1 DiscussionOften this accumulation is accompa-nied
13、 by deposition of organic and inorganic material.3.2.9 microbicides, na physical or chemical agent thatkills microorganisms.1This practice is under the jurisdiction of ASTM Committee E35 on Pesticides,Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.
14、15 on Antimicrobial Agents.Current edition approved April 1, 2013. Published May 2013. Originallyapproved in 1978. Last previous edition approved in 2007 as E645 07. DOI:10.1520/E0645-13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at servicea
15、stm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19
16、428-2959. United States14. Summary of Practice4.1 Microbicides are evaluated against microbes under con-ditions simulating a cooling water system. Microbicides atconcentrations that are expected to control the microbes areadded to cooling water.At selected time periods, the number ofmicrobes or meas
17、urable component of the microbes are deter-mined and compared to values at the start of the experiment.Bacteria (aerobic and anaerobic), fungi, and algae may bedetected by a number of methods, such as plate counting, MostProbable Number (MPN), chlorophyll content, adenosine-5-triphosphate (ATP). The
18、 investigator will determine the rangeof microbicide concentration for acceptable efficacy basedupon laboratory testing that may be used to satisfy registrationor customer needs.5. Significance and Use5.1 This practice determines potentially effective microbi-cides for use in cooling water systems u
19、sing cooling water anddeposits/biofilm obtained from the field. The addition ofdeposits/biofilms addresses the need to include the majorsource of microorganisms in cooling water systems. Even withthis addition, laboratory results may not be totally predictive ofmicrobicidal effectiveness in the fiel
20、d. This is because condi-tions in the field affecting microbicide effectiveness are diffi-cult to mimic in the laboratory. These conditions that affectmicrobicide efficacy include blow-down rate, addition ofmakeup water, water hardness, hydrocarbon leaks, pH, sedi-ment loading, dissolved solids, mic
21、robes in slime (biofilms),and deposits (salts, iron minerals, organics, and so forth) onsurfaces. An additional factor is the difficulty in enumeratingall microbes in the water due to the lack of adequate recoverymedia. Guidelines that address formation of and testing forsurface-attached microbes (b
22、iofilms) may be found in GuideE1427, while a guideline for unconventional measurement ofmicrobes is found in Guide E1326.6. Apparatus6.1 Balancea calibrated analytical balance sensitive to0.1 mg to weigh the candidate microbicide for preparation ofstock solutions.6.2 Containersflasks, bottles, or te
23、st tubes suitable forshaking shall be sterile for use.6.3 Colony Countersmanual, such as Quebec, Buck, orWolffhuegel, or a proven colony image analyzer (electronic/scanner type) are suitable for counting plates after incubation.6.4 Spiral Plater (alternative).6.5 Constant Temperature Shakera reliabl
24、e constant-temperature shaker 62C (water bath or incubator shaker) toprovide mixing and aeration and to maintain temperatureduring the contact period at a setting within the temperaturerange selected in 10.2.6.6 Petri Dishes, sterile, 100 by 15-mm plastic or borosili-cate glass.6.7 Pipettesstandard
25、pipettes, sterile, with appropriatecalibrations, or other suitable delivery systems, such micropi-petters.6.8 Sterilizerspressurized steam sterilizer (for media,containers, and so forth), hot air oven for containers, and filterapparatus for filter sterilization (disposable filter units, 250 mL,0.22-
26、m pore size).6.9 Stirrerrequired to mix the cooling water sample whileit is being dispensed into test containers. This can be amagnetic stirrer, a propeller-type stirrer, or any other suitabledevice.6.10 Volumetric Flasks, 100 mL, are convenient for prepar-ing microbicide stock solutions. Smaller vo
27、lume flasks may beused where appropriate.6.11 Blendera blender, stomacher, sonic bath, or vortexmixer to homogenize the microbial deposit before mixing itwith the cooling water.6.12 Microscope, providing a magnification range of 400 to1000 with a suitable light source. Phase contrast or dark-fieldca
28、pability may be necessary.6.13 Filter apparatus, with 0.2 m filter.7. Reagents and Materials7.1 Purity of ReagentsThe principal reagent used is water,but other solvents may be necessary in preparing the microbi-cide stock solutions. Reagent grade organic solvents arenormally used if water is not a s
29、uitable diluent for dissolving amicrobicide. If a solvent is used, an additional control must beperformed that has solvent without any microbicide added tothe cooling water sample. This is used to demonstrate that thesolvent has no appreciable effect on the test results.7.2 Purity of WaterAll refere
30、nce to water as a diluent orreagent shall mean distilled water or water of equal purity,unless otherwise noted.7.3 Culture Media:7.3.1 A general bacterial agar medium, such as glucoseextract agar, tryptic soy agar, R2A agar, or dry film is used forconducting bacterial counts on test samples. Other m
31、edia, suchas selective or differential types (that is, for the quantificationof sulfate-reducing bacteria,Test Methods D4412) may be usedfor detecting desired bacteria. MPN or ATP measurement mayalso be used to quantify the bacteria (Guide E1326). Once aspecific agar medium or other method of measur
32、ement ischosen, it must be used throughout this procedure.7.3.2 A general fungal medium, such as an inhibitory moldagar or Sabouraud dextrose agar, is used for conducting fungalcounts on the samples. This medium must be able to inhibit thegrowth of bacteria.7.3.3 Bristols medium,4or a suitable equiv
33、alent, is therecommended medium for the growth of algae.7.4 Dilution Water BlanksSterile, 99 or 9-mL phosphatebuffered saline or phosphate buffered magnesium chloridedilution blanks are convenient for diluting test samples forviable counts. Buffer strength and salinity can be adjusted tomimic experi
34、mental or field conditions.4Starr, R. C., and Zeikus, J. A., “The Culture Collection of Algae at theUniversity of Texas at Austin,” Journal of Psychology, Vol 23, No. 5, 1987, pp.147.E645 1327.4.1 Phosphate Buffered Dilution Water Blanks.7.4.1.1 Phosphate Buffer Solution, StockDissolve 34.0 gof pota
35、ssium dihydrogen phosphate (KH2PO4) in 500 mL ofwater.Adjust pH to 7.2 6 0.2 with NaOH solution (40 g/L) andbring to 1000 mL with water. Sterilize by filtration or auto-clave.7.4.1.2 Phosphate Buffered Saline Dilution WaterAdd1.25 mL of stock phosphate buffer solution and 8.75 g of NaClto a volumetr
36、ic flask, fill with reagent water to the 1000-mLmark, and mix. Final pH should be 7.2 6 0.2. Dispense inamount that will provide 99 6 2mLor96 1 mL aftersterilization into screw-cap dilution bottles or tubes. Sterilizeimmediately.7.4.2 Phosphate Buffered Magnesium Chloride DilutionWaterAdd 1.25 mL of
37、 stock phosphate buffer solution and5.0 mL of magnesium chloride solution (81.1 g MgCl26H2O/L, reagent grade water) to 1000 mL of water. Adjust pHto 7.2 6 0.2. Dispense in amounts that will provide 99 6 2mLor 9 6 1 mL after sterilization into screw-cap dilution bottlesor tubes. Sterilize immediately
38、.7.5 Cooling Water Sample:7.5.1 The cooling water sample will be collected in a sterilecontainer (1-gal or 2.2-L plastic bottles are convenient). Thetemperature and pH should be determined at the time of samplecollection. The presence of additives in the cooling tower watermay affect the effectivene
39、ss of the microbicides, therefore, ahistory of the samples should be obtained or analysis of thewater for additives should be conducted. Stop biocide additionat least 4 h before collection of samples, or an appropriatebiocide inactivator must be added to the sample. Do not exposesamples to temperatu
40、re extremes during transit. If a variationof 1.0 pH unit exists between the time of sampling and testing,the sample should be discarded. The test procedure should beinitiated within 24 h after collection. Samples received fromthe field must be refrigerated (4 6 2C).7.5.2 Collect deposits of microbia
41、l composition in sterilecontainers from any affected areas of the cooling tower, such asthe distribution deck, slats, or sump area. Transport the depositsamples with the water sample following the same precautions.Upon receipt at the laboratory, conduct microscopic examina-tion of the deposits to co
42、nfirm that they are microbiological innature. If testing for algicidal or fungicidal activity, or both, thesample must contain algae or fungi, or both.8. Preparation of the Test Samples8.1 The cooling water sample may be used as received orinoculated with known microorganisms. If the water is usedon
43、ly as a substrate and known microorganisms5will be addedas inoculum, the water should be filter-sterilized (using a0.2 m filter system) prior to the addition of microorganisms. Ifa biofilm sample or microbiological deposit is available, it maybe used as the inoculum in either filtered or non-filtere
44、dsterilized cooling water. The biolfilm or slime must behomogenized/disaggregated so that no clumps are present. Thiscan be accomplished by vortexing, sonicating, or any othermethod that disperses the clumps. No more than 10 % of thetotal weight (w/v) of the samples should be biofilm or deposit.A sy
45、nthetic cooling water may also be used as the samplewater.8.2 Place the cooling water sample on a stirrer and mixcontinuously. Transfer 100 6 2 mL (or 100 6 2 g) to sterileflasks or bottles. Prepare at least duplicate flasks or bottles foreach microbicide concentration to be tested. In addition,prep
46、are duplicate controls to which no microbicide will beadded. If a solvent other than water is used to make themicrobicide stock solutions, also include solvent controlbottles that contain as much of the solvent as is added to themicrobicide test containers (see 8.1). The 100-mL watervolume is a stan
47、dard volume used in all previously publishedevaluation of cooling water microbicides. However, othervolumes of cooling water may be used in this test.8.3 After the test aliquots have been transferred to flasks,determine the viable count of microorganisms in the controlflask in accordance with standa
48、rd microbiological methods.Suggested dilutions for this are 102,103,104, and 105.Bacterial numbers should be at least 105bacteria/mL. Algaeand fungal numbers are determined by pour-plate, spread-plate,or MPN. A minimum of 103algal CFU/mL and/or fungalCFU/mL is necessary to conduct the effectiveness
49、test againstalgae or fungi in cooling water. Obtaining these numbers ofalgae and fungi may require addition of algae, fungi or biofilm.Other microbial detection methods may be used (see GuideE1326). Initial or baseline values must be established for thesemethods.9. Preparation of Microbicide Stock Solutions9.1 The appropriate test concentrations for a particularmicrobicide must be determined by the investigator. Usually,microbicide concentrations depend upon the biocide, itsapplication, and its registration range. For oxidizing biocides, itis i
