1、Designation: E645 13E645 18Standard 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 rev
2、ision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope Scope*1.1 This practice outlines a procedure for evaluating the efficacy of microbicides (algicides, bactericides, and fungicides
3、) thatwill be used for controlling microbial growth in cooling water systems. The microbicides will be evaluated using simulated or realcooling tower water against (1) microbes from cooling water, (2) microbes in microbiological deposits (biofilms) from operatingcooling systems, or (3) microorganism
4、s known to contaminate cooling water systems, or a combination thereof. This practiceshould be performed by individuals familiar with microbiological techniques.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 This stan
5、dard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1
6、.4 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Tra
7、de (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D3731 Practices for Measurement of Chlorophyll Content of Algae in Surface WatersD4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in WaterD4412 Test Methods for Sulfate-Reducing Bacteria in Water and Water-Form
8、ed DepositsE1054 Test Methods for Evaluation of Inactivators of Antimicrobial AgentsE1326 Guide for Evaluating Non-culture Microbiological TestsE1427 Guide for Selecting Test Methods to Determine the Effectiveness of Antimicrobial Agents and Other Chemicals for thePrevention, Inactivation and Remova
9、l of Biofilm (Withdrawn 2009)3E2756 Terminology Relating to Antimicrobial and Antiviral Agents3. Terminology3.1 For definitions of terms used in this practice, see Terminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 algicide, na chemical agent that kills algae; unicellular or
10、filamentous chlorophyll-containing plants.3.2.2 bactericide, na physical or chemical agent that kills bacteria, but not necessarily bacterial spores.3.2.3 biofilm, na dynamic, self-organized accumulation of microorganisms and environmental by-products immobilized on asubstrate and embedded in an org
11、anic polymer matrix.3.2.4 cooling system, nequipment and coolant used for the removal of heat from processes, equipment, or both.3.2.4.1 Discussion1 This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsib
12、ility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2013Oct. 1, 2018. Published May 2013October 2018. Originally approved in 1978. Last previous edition approved in 20072013 asE645 07.E645 13. DOI: 10.1520/E0645-13.10.1520/E0645-18.2 For referencedASTM standards, vi
13、sit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 The last approved version of this historical standard is referenced on www.astm.org.This docu
14、ment is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as
15、appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United
16、 States1The most common medium used for removal or transfer of heat is water. The heated water then can be discharged into a receivingbody (once through cooling system) or it can be cooled and reused (recirculating cooling system).3.2.5 cooling tower, na structure used to dissipate heat in open reci
17、rculating cooling systems.3.2.6 cooling water, nany water-based solution that absorbs and transfers heat in a heat exchange system.3.2.7 fungicides, na physical or chemical agent that kills fungi; that is, vegetative mycelia and/or budding yeasts includingspores and/or conidia.3.2.8 microbial biofou
18、ling, nthe unwanted accumulation of bacterial, fungal, or algal cells, or any combination thereof andtheir products on surfaces.3.2.8.1 DiscussionOften this accumulation is accompanied by deposition of organic and inorganic material.3.2.9 microbicides, na physical or chemical agent that kills microo
19、rganisms.4. Summary of Practice4.1 Microbicides are evaluated against microbes under conditions simulating a cooling water system. Microbicides atconcentrations that are expected to control the microbes are added to cooling water. At selected time periods, the number ofmicrobes or measurable compone
20、nt of the microbes are determined and compared to values at the start of the experiment. Bacteria(aerobic and anaerobic), fungi, and algae may be detected by a number of methods, such as plate counting, Most Probable Number(MPN), chlorophyll content, adenosine-5-triphosphate (ATP). The investigator
21、will determine the range of microbicideconcentration for acceptable efficacy based upon laboratory testing that may be used to satisfy registration or customer needs.5. Significance and Use5.1 This practice determines potentially effective microbicides for use in cooling water systems using cooling
22、water anddeposits/biofilm obtained from the field. The addition of deposits/biofilms addresses the need to include the major source ofmicroorganisms in cooling water systems. Even with this addition, laboratory results may not be totally predictive of microbicidaleffectiveness in the field. This is
23、because conditions in the field affecting microbicide effectiveness are difficult to mimic in thelaboratory. These conditions that affect microbicide efficacy include blow-down rate, addition of makeup water, water hardness,hydrocarbon leaks, pH, sediment loading, dissolved solids, microbes in slime
24、 (biofilms), and deposits (salts, iron minerals,organics, and so forth) on surfaces. An additional factor is the difficulty in enumerating all microbes in the water due to the lackof adequate recovery media. Guidelines that address formation of and testing for surface-attached microbes (biofilms) ma
25、y befound in Guide E1427, while a guideline for unconventional measurement of microbes is found in Guide E1326.6. Apparatus6.1 Balancea calibrated analytical balance sensitive to 0.1 mg to weigh the candidate microbicide for preparation of stocksolutions.6.2 Containersflasks, bottles, or test tubes
26、suitable for shaking shall be sterile for use.6.3 Colony Countersmanual, such as Quebec, Buck, or Wolffhuegel, or a proven colony image analyzer (electronic/scannertype) are suitable for counting plates after incubation.6.4 Spiral Plater (alternative).6.5 Constant Temperature Shakera reliable consta
27、nt-temperature shaker 62C62 C (water bath or incubator shaker) toprovide mixing and aeration and to maintain temperature during the contact period at a setting within the temperature rangeselected in 10.2.6.6 Petri Dishes, sterile, 100 by 15-mm plastic or borosilicate glass.6.7 Pipettesstandard pipe
28、ttes, sterile, with appropriate calibrations, or other suitable delivery systems, such micropipetters.6.8 Sterilizerspressurized steam sterilizer (for media, containers, and so forth), hot air oven for containers, and filter apparatusfor filter sterilization (disposable filter units, 250 mL, 0.22-m
29、pore size).6.9 Stirrerrequired to mix the cooling water sample while it is being dispensed into test containers. This can be a magneticstirrer, a propeller-type stirrer, or any other suitable device.6.10 Volumetric Flasks, 100 mL, are convenient for preparing microbicide stock solutions. Smaller vol
30、ume flasks may be usedwhere appropriate.6.11 Blendera blender, stomacher, sonic bath, or vortex mixer to homogenize the microbial deposit before mixing it with thecooling water.E645 1826.12 Microscope, providing a magnification range of 400 to 1000 with a suitable light source. Phase contrast or dar
31、k-fieldcapability 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 microbicidestock solutions. Reagent grade organic solvents are normally used if water i
32、s not a suitable diluent for dissolving a microbicide.If a solvent is used, an additional control must be performed that has solvent without any microbicide added to the cooling watersample. This is used to demonstrate that the solvent has no appreciable effect on the test results.7.2 Purity of Wate
33、rAll reference to water as a diluent or reagent shall mean distilled water or water of equal purity, unlessotherwise noted.7.3 Culture Media:7.3.1 A general bacterial agar medium, such as glucose extract agar, tryptic soy agar, R2A agar, or dry film is used forconducting bacterial counts on test sam
34、ples. Other media, such as selective or differential types (that is, for the quantification ofsulfate-reducing bacteria, Test Methods D4412) may be used for detecting desired bacteria. MPN or ATP (Test Method D4012)measurement may also be used to quantify the bacteriabioburdens (Guide E1326). Once a
35、 specific agar medium or other methodof measurement is chosen, it must be used throughout this procedure.7.3.2 A general fungal medium, such as an inhibitory mold agar or Sabouraud dextrose agar, is used for conducting fungalcounts on the samples. This medium must be able to inhibit the growth of ba
36、cteria.7.3.3 Bristols medium,4 or a suitable equivalent, is the recommended medium for the growth of algae.7.4 Dilution Water BlanksSterile, 99 or 9-mL phosphate buffered saline or phosphate buffered magnesium chloride dilutionblanks are convenient for diluting test samples for viable counts. Buffer
37、 strength and salinity can be adjusted to mimic experimentalor field conditions.7.4.1 Phosphate Buffered Dilution Water Blanks.7.4.1.1 Phosphate Buffer Solution, StockDissolve 34.0 g of potassium dihydrogen phosphate (KH2PO4 ) in 500 mL of water.Adjust pH to 7.2 6 0.2 with NaOH solution (40 g/L) and
38、 bring to 1000 mL with water. Sterilize by filtration or autoclave.7.4.1.2 Phosphate Buffered Saline Dilution WaterAdd 1.25 mL of stock phosphate buffer solution and 8.75 g of NaCl to avolumetric flask, fill with reagent water to the 1000-mL mark, and mix. Final pH should be 7.2 6 0.2. Dispense in a
39、mount thatwill provide 99 6 2 mL or 9 6 1 mL after sterilization into screw-cap dilution bottles or tubes. Sterilize immediately.7.4.2 Phosphate Buffered Magnesium Chloride Dilution WaterAdd 1.25 mL of stock phosphate buffer solution and 5.0 mLof magnesium chloride solution (81.1 g MgCl2 6 H2O/L, re
40、agent grade water) to 1000 mL of water. Adjust pH to 7.2 6 0.2.Dispense in amounts that will provide 99 6 2 mL or 9 6 1 mL after sterilization into screw-cap dilution bottles or tubes. Sterilizeimmediately.7.5 Cooling Water Sample:7.5.1 The cooling water sample will be collected in a sterile contain
41、er (1-gal or 2.2-L plastic bottles are convenient). Thetemperature and pH should be determined at the time of sample collection. The presence of additives in the cooling tower watermay affect the effectiveness of the microbicides, therefore, a history of the samples should be obtained or analysis of
42、 the water foradditives should be conducted. Stop biocide addition at least 4 h before collection of samples, or an appropriate biocide inactivatormust be added to the sample. Do not expose samples to temperature extremes during transit. If a variation of 1.0 pH unit existsbetween the time of sampli
43、ng and testing, the sample should be discarded. The test procedure should be initiated within 24 h aftercollection. Samples received from the field must be refrigerated (4 6 2C).(4 6 2 C).7.5.2 Collect deposits of microbial composition in sterile containers from any affected areas of the cooling tow
44、er, such as thedistribution deck, slats, or sump area. Transport the deposit samples with the water sample following the same precautions. Uponreceipt at the laboratory, conduct microscopic examination of the deposits to confirm that they are microbiological in nature. Iftesting for algicidal or fun
45、gicidal activity, or both, the sample must contain algae or fungi, or both.8. Preparation of the Test Samples8.1 The cooling water sample may be used as received or inoculated with known microorganisms. If the water is used only asa substrate and known microorganisms5 will be added as inoculum, the
46、water should be filter-sterilized (using a 0.2 m filtersystem) prior to the addition of microorganisms. If a biofilm sample or microbiological deposit is available, it may be used as theinoculum in either filtered or non-filtered sterilized cooling water. The biolfilm or slime must be homogenized/di
47、saggregated sothat no clumps are present. This can be accomplished by vortexing, sonicating, or any other method that disperses the clumps. Nomore than 10 % of the total weight (w/v) of the samples should be biofilm or deposit. A synthetic cooling water may also be usedas the sample water.4 Starr, R
48、. C., and Zeikus, J. A., “The Culture Collection of Algae at the University of Texas at Austin,” Journal of Psychology, Vol 23, No. 5, 1987, pp. 147.5 Pesticide Assessment Guidelines, Subdivision G, Product Performance, U.S. Environmental Protection Agency, November 1982, Section 92.4, or most curre
49、nt edition.E645 1838.2 Place the cooling water sample on a stirrer and mix continuously. Transfer 100 6 2 mL (or 100 6 2 g) to sterile flasks orbottles. Prepare at least duplicate flasks or bottles for each microbicide concentration to be tested. In addition, prepare duplicatecontrols to which no microbicide will be added. If a solvent other than water is used to make the microbicide stock solutions, alsoinclude solvent control bottles that contain as much of the solvent as is added to the microbicide test containers (see 8.1). The100-mL w
