1、Designation: E 645 07Standard Test Method forEfficacy of Microbicides Used in Cooling Water Systems1This standard is issued under the fixed designation E 645; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revisio
2、n. 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 This test method outlines a procedure for evaluating theefficacy of microbicides (algicides, bactericides, and fungi-cides) that
3、will be 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 t
4、o contaminate cooling water systems,or a combination thereof. This test method should be per-formed by individuals familiar with microbiological tech-niques.1.2 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user o
5、f 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:2D 3731 Practices for Measurement of Chlorophyll Contentof Algae in Surface WatersD 4012 Test Method for Adenosine T
6、riphosphate (ATP)Content of Microorganisms in WaterD 4412 Test Methods for Sulfate-Reducing Bacteria inWater and Water-Formed DepositsE 1054 Test Methods for Evaluation of Inactivators ofAntimicrobial AgentsE 1326 Guide for Evaluating Nonconventional Microbio-logical Tests Used for Enumerating Bacte
7、riaE 1427 Guide for Selecting Test Methods to Determine theEffectiveness of Antimicrobial Agents and Other Chemi-cals for the Prevention, Inactivation and Removal ofBiofilm3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 algicide, na chemical agent that kills algae; unicel-lula
8、r chlorophyll-containing plants.3.1.2 bactericides, na physical or chemical agent thatkills bacteria, but not necessarily bacterial spores.3.1.3 biofilm, na dynamic, self-organized accumulation ofmicroorganisms and environmental by-products immobilizedon a substrate and embedded in an organic polyme
9、r matrix.3.1.4 cooling system, nan assemblage of equipment forthe removal of heat from processes or equipment, or both. Themost common medium used for removal or transfer of heat iswater. The heated water then can be discharged into a receivingbody (once through cooling system) or it can be cooled a
10、ndreused (recirculating cooling system).3.1.5 cooling tower, na structure used to dissipate heat inopen recirculating cooling systems.3.1.6 cooling water, nmedium used to transfer heat incooling systems.3.1.7 fungicides, na physical or chemical agent that killsfungi; that is, vegetative mycelia and/
11、or budding yeasts includ-ing spores and/or conidia.3.1.8 microbial biofouling, nthe unwanted accumulationof bacterial, fungal or algal cells, or any combination thereofand their products on surfaces.Often this accumulation is accompanied by deposition oforganic and inorganic material.3.1.9 microbici
12、des, na physical or chemical agent thatkills microorganisms.4. Summary of Test Method4.1 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
13、 periods, the number ofmicrobes or measurable 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, Most1This test method is under the jurisdict
14、ion of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2007. Published April 2007. Originallyapproved in 1978. Last previous edition approved in 2002 as E 645 02a.2For refe
15、renced 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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, Wes
16、t Conshohocken, PA 19428-2959, United States.Probable Number (MPN), chlorophyll content, adenosine-58-triphosphate (ATP). The investigator will determine the rangeof microbicide concentration for acceptable efficacy basedupon laboratory testing that may be used to satisfy registrationneeds.5. Signif
17、icance and Use5.1 This test method determines potentially effective micro-bicides for use in cooling water systems using cooling waterand deposits/biofilm obtained from the field. The addition ofdeposits/biofilms addresses the need to include the majorsource of microorganisms in cooling water system
18、s. Even withthis addition, laboratory results may not be totally predictive ofmicrobicidal effectiveness in the field. This is because condi-tions in the field affecting microbicide effectiveness are diffi-cult to mimic in the laboratory. These include blow down rate,addition of makeup water, water
19、hardness, hydrocarbon leaks,pH, sediment loading, dissolved solids, microbes in slime, anddeposits (biofilms) on surfaces. An additional factor is thedifficulty in enumerating all microbes in the water due to thelack of adequate recovery media. Guidelines that addressformation of and testing for sur
20、face-attached microbes (bio-films) may be found in Guides E 1427, while a guideline forunconventional measurement of microbes is found in GuideE 1326.6. Apparatus6.1 BalanceA calibrated analytical balance sensitive to0.1 mg to weigh the candidate microbicide for preparation ofstock solutions.6.2 Con
21、tainersFlasks, bottles, or test tubes suitable forshaking shall be sterilized prior to 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
22、Constant Temperature ShakerA reliable 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 boros
23、ili-cate glass.6.7 PipettesStandard 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 (d
24、isposable filter units, 250 mL,0.22-m pore size).6.9 StirrerA stirrer is required to mix the cooling watersample while it is being dispensed into test containers. This canbe a magnetic stirrer, a propeller-type stirrer, or any othersuitable device.6.10 Volumetric Flasks, 100 mL, are convenient for p
25、repar-ing microbicide stock solutions. Smaller volume 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, provides a magnification of 400 to 10003and is complete w
26、ith a suitable light source. Phase contrast ordark-field capability 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
27、grade organic solvents arenormally used if water is not a suitable 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 appreciab
28、le effect on the test results.7.2 Purity of WaterAll reference 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 GlucoseExtractAgar, Tryptic SoyAgar, R2AAgar is used for conduct-
29、ing bacterial counts on test samples. Other media, such asselective or differential types may be used for detecting desiredbacteria. ATP measurement may also be used to quantify thebacteria.7.3.2 A general fungal medium, such as an inhibitory moldagar, Sabouraud Dextrose Agar, is used for conducting
30、 fungalcounts on the samples. This medium must be able to inhibit thegrowth of bacteria.7.3.3 Bristols medium,3or a suitable equivalent, is therecommended medium for the growth of algae.7.4 Dilution Water BlanksSterile, 99 or 9-mL phosphatebuffered saline or phosphate buffered magnesium chloridedilu
31、tion blanks are convenient for diluting test samples forviable counts. Buffer strength and salinity can be adjusted tomimic experimental or field conditions.7.4.1 Phosphate Buffered Dilution Water Blanks.7.4.1.1 Phosphate Buffer Solution, StockDissolve 34.0 gof potassium dihydrogen phosphate (KH2PO4
32、) 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 volumetric flask, fill with reagent water
33、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 stock phosphate buffer solution a
34、nd5.0 mL of magnesium chloride solution (81.1 g MgCl26H2O/L, reagent grade water) to 1000 mL of water. Adjust pH3Starr, R. C., and Zeikus, J. A., “The Culture Collection of Algae at theUniversity of Texas at Austin,” Journal of Psychology, Vol 23(5): pp. 147, 1987.E645072to 7.2 6 0.2. Dispense in am
35、ount that will provide 99 6 2mLor 9 6 1 mL after sterilization into screw-cap dilution bottlesor tubes. Sterilize immediately.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
36、be determined at the time of samplecollection. The presence of additives in the cooling tower watermay affect the effectiveness 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 befo
37、re collection of samples, or an appropriatebiocide inactivator must be added to the sample. Do not exposesamples to temperature 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
38、within 24 h after collection.7.5.2 Collect deposits of microbial 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 lab
39、oratory, conduct microscopic examina-tion of the deposits to confirm that it is microbiological innature. If testing for algicidal or fungicide 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 ori
40、noculated with known microorganisms. If the water is usedonly as a substrate and known microorganisms4will be addedas inoculum, the water should be filter-sterilized (using a 0.2m filter system) prior to the addition of microorganisms. If abiofilm sample or microbiological deposit is available, it m
41、aybe used as the inoculum in either filtered or non-filteredsterilized cooling water. No more than 10 % of the total weight(w/v) of the samples should be biofilm or deposit. A syntheticcooling water may also be used as the sample water.8.2 Place the cooling water sample on a stirrer and mixedcontinu
42、ously. 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,prepare duplicate controls to which no microbicide will beadded. If a solvent other than water is used to make themicrobicid
43、e stock solutions, also include solvent controlbottles that contain as much of the solvent as is added to themicrobicide test containers (see 8.1).8.3 After the test aliquots have been transferred to flasks,determine the viable count of microorganisms in the controlflask in accordance with standard
44、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.Aminimum of 103algae CFU/mLof cooling water or103fungi CFU/mLof cooling water is necessary to c
45、onduct theeffectiveness test against algae or fungi. Obtaining thesenumbers of algae and fungi may require addition of algae,fungi or biofilm. Other microbial detection methods may beused (see Guide E 1326). Initial or baseline values must beestablished for these methods.9. Preparation of Microbicid
46、e Stock Solutions9.1 The appropriate test concentrations for a particularmicrobicide must be determined by the investigator. Usually,microbicide concentrations are in the range from 1 to 50 mgactive/L. For this range, concentrations of 1.0, 2.0, 5.0, 10.0,25, and 50 mg active/L would be tested. It i
47、s convenient,therefore, to make two microbicide stock solutions. The firstshould be 5.0 mg active/mL and the second should bea1to10dilution of the first stock solution. The first stock solution isused to make the 10.0, 25.0, and 50.0-mg active/L testconcentrations, by addition of 0.2, 0.5, and 1.0 m
48、L, respec-tively, per 100 mLof sample. The second stock is used to makethe more dilute test concentrations. Using this method, thevolume of microbicide stock solution added will not exceed1 % of the total volume of the cooling water aliquots. Thisrelative volume is an important consideration, partic
49、ularlywhen solvents other than water are used to make the microbi-cide stock solutions. If an acceptable reduction in microbialnumbers is not achieved with these concentrations, the inves-tigator must make the appropriate adjustments in the microbi-cide stock solutions and in the selected test concentrations.Microbicide test stock solutions should be prepared no morethan 3 h before the test.9.2 The initial microbicide stock solution is prepared byweighing the microbicide on an analytical balance, transferringit to a volumetric flask, and bringing it t
