1、Designation: E 2275 03e1Standard Practice forEvaluating Water-Miscible Metalworking Fluid Bioresistanceand Antimicrobial Pesticide Performance1This standard is issued under the fixed designation E 2275; the number immediately following the designation indicates the year oforiginal adoption or, in th
2、e 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.e1NOTEFootnote 1 was editorially revised in November 2003.1. Scope1.1 This practice addresses the
3、 evaluation of the relativeinherent bioresistance of water-miscible metalworking fluids,the bioresistance attributable to augmentation with antimicro-bial pesticides or both. It replaces Methods D 3946 and E 686.1.2 In this practice relative bioresistance is determined bychallenging metalworking flu
4、ids with a biological inoculumthat may either be characterized (comprised of one or moreknown biological cultures) or uncharacterized (comprised ofbiologically contaminated metalworking fluid or one or moreunidentified isolates from deteriorated metalworking fluid).Challenged fluid bioresistance is
5、defined in terms of resistanceto biomass increase, viable cell recovery increase, chemicalproperty change, physical property change or some combina-tion thereof.1.3 This practice is applicable to antimicrobial agents thatare incorporated into either the metalworking fluid concentrateor end-use dilut
6、ion. It is also applicable to metalworking fluidsthat are formulated using non-microbicidal, inherently biore-sistant components.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish
7、 appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 888 Test Methods for Dissolved Oxygen in WaterD 1067 Test Methods for Acidity or Alkalinity of WaterD 1193 Specification for Reagent Grade Wa
8、terD 3342 Method for Dispersion Stability of New (Unused)Rolling Oil Dispersions in WaterD 3519 Test Method for Foam in Aqueous Media (BlenderTest)D 3601 Test Method for Foam in Aqueous Media (BottleTest)D 4012 Test Method for Adenosine Triphosphate (ATP)Content of Microorganisms in WaterD 4627 Meth
9、od for Iron Chip Corrosion for Water-Dilutable Metalworking FluidsD 5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating MethodsE70 Test Method for pH of Aqueous Solutions with theGlass ElectrodeE 1326 Guide for Evaluating Nonconventional Microbio-logical tests Used f
10、or Enumerating BacteriaE 2169 Practice for Selecting Antimicrobial Pesticides foruse in Water-miscible Metalworking Fluids2.2 Other Standards:4.027 Synthetic Hard Water39215A.6a Heterotrophic Plate Count Media, Plate CountAgar49216 Direct Total Microbial Count4Microbiological Test 52.3 Government St
11、andard:40 CFR 156 Labeling Requirements for Pesticides and De-vices3. Terminology3.1 Definitions:3.1.1 active ingredient, nthe chemical component or com-ponents of an antimicrobial pesticide that provides its micro-bicidal performance.3.1.2 antimicrobial pesticide, nchemical additive regis-tered und
12、er 40 CFR 152, for use to inhibit growth, proliferationor both of microorganisms.1This practice is under the jurisdiction of ASTM Committee E35 on Pesticidesand Alternative Control Agents and is the direct responsibility of SubcommitteeE35.15 on Antimicrobial Agents.Current edition approved April 10
13、, 2003. Published May 2003.2For 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.3AOAC International Methods of Ana
14、lysis, AOAC International, Gaithersburg,MD.4Available from American Public Health Association (APHA) Standard Meth-ods for the Examination of Water and Wastewater 800 I Street, NW Washington, DC20001.5Available from U.S. Pharmacopeia/National Formulary (USP/NF), 12601Twinbrook Parkway Rockville, MD
15、20852.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.3 as supplied, adjantimicrobial pesticide finishedproduct including the active ingredient(s), solvent and anyadditional inactive ingredients.3.1.4 biocide, nany chemical inten
16、ded for use to killorganisms.3.1.5 bioresistant, adjability to withstand biological at-tack.3.1.5.1 DiscussionBioresistant, or recalcitrant, chemicalsare not readily metabolized by microorganisms.3.1.6 biostatic, adjable to prevent existing microbial con-taminants from growing or proliferating, but
17、unable to killthem.3.1.6.1 DiscussionBiostatic additives may be registeredantimicrobial pesticides or unregistered chemicals with otherperformance properties. The difference between biocidal andbiostatic performance may be attributed to dose, chemistry orboth.3.1.7 dose, nconcentration of antimicrob
18、ial pesticideadded to treated solution.3.1.7.1 DiscussionDose is generally expressed as eitherppm active ingredient (a.i.) or ppm as supplied (a.s.).3.1.8 inactive ingredient, ncomponent of antimicrobialpesticide that is not directly responsible for the pesticidesantimicrobial performance.3.1.8.1 Di
19、scussionInactive ingredients may include, butare not limited to solvents and chemicals that improve thepesticides non-biocidal performance properties, such as mis-cibility and reactivity with non-target molecules in the treatedmaterial.3.1.9 minimum inhibitory concentration (MIC), nlowesttreatment-d
20、ose that will prevent test population from growing,proliferating or otherwise contributing to biodeterioration.3.2 Abbreviations:3.2.1 a.i.active ingredient3.2.2 a.s.as supplied3.2.3 ATCCamerican type culture collection3.2.4 CFUcolony forming unit4. Summary of Practice4.1 End-use dilutions of one or
21、 more water-miscible metal-working fluids are dispensed into microcosms. The fluids maybe fresh or aged, dosed with one or more antimicrobialpesticides or undosed. Microcosms are challenged with eitheruncharacterized or characterized biological inocula. After in-oculation, microcosms are aerated eit
22、her continuously or peri-odically to simulate recirculation conditions in coolant sys-tems. Chips may also be added to microcosms to simulate chipaccumulation in coolant systems.4.2 After inoculation, fluid samples are drawn from eachmicrocosm periodically and tested for the parameters ofinterest, i
23、ncluding but not limited to microbial viable counts.Depending on the test objectives, the test duration may rangefrom 24 h to three months.4.2.1 Shorter test periods are used to evaluate microbicidespeed of kill and metalworking formulation initial bioresis-tance.4.2.2 Longer test periods are used t
24、o evaluate metalworkingfluid formulation resistance to repeated challenges. For testslasting longer than one-week, 10 to 80 % of the fluid isexchanged weekly with fresh fluid before the additionalchallenge. The percentage of fluid exchange should reflectanticipated fluid turnover rates in fluids end
25、-use application.4.3 Bioresistance is determined as the test fluids relativeability to prevent the proliferation of challenge microbes, retainits original chemical or physical properties of some combina-tion of the above. The bioresistance of test formulations isdefined relative to that of a benchma
26、rk or control formulation.5. Significance and Use5.1 This practice provides laboratory procedures for ratingthe relative bioresistance of metalworking fluid formulations,for determining the need for microbicide addition prior to orduring fluid use in metalworking systems and for evaluatingmicrobicid
27、e performance. General considerations for microbi-cide selection are provided in Practice E 2169.5.2 The factors affecting challenge population numbers,taxonomic diversity, physiological state, inoculation frequencyand biodeterioration effects in recirculating metalworking fluidsystems are varied an
28、d only partially understood. Conse-quently, the results of tests completed in accordance with thispractice should be used only to compare the relative perfor-mance of products or microbicide treatments included in a testseries. Results should not be construed as predicting actualfield performance.6.
29、 Apparatus6.1 Air Supply, air provided at no more than 110 kPa.NOTE 1Any air source that is free of organic vapors, organic matteror other objectionable material may be used. Sterile air need not be usedfor the uncharacterized inoculum, but shall be used for the characterizedinoculum. If necessary,
30、air may be sterilized either by inserting, in series,two commercially available in-line sterile filters designed for this purpose.Alternatively an in-line filter may be prepared as follows: Pack two 150mm long drying tubes (bulb-type) loosely with borosilicate glass wool inseries with neoprene stopp
31、ers, glass tubing and neoprene tubing. Wraploosely in aluminum foil and steam sterilize at 103 to 138 kPa (15 to 20psi) for 30 min or dry heat sterilize at 160C for 2 h. Cool to roomtemperature while wrapped. Insert into air line with bulbs on upstreamside. Whether using a commercial or fabricated f
32、ilter, average lifetime incontinuous use is two weeks. Discard sooner if upstream filter becomeswet or contaminated with oil.6.2 Aquarium Tubing, 6.35 mm (0.25 in.) diameter, siliconeor vinyl.6.3 Autoclave, with both steam cycle (80 to 100C) andsterilization cycle (15 min at $ 121C) capability.6.4 A
33、djustable Volume Pipetters, with sterile disposabletips. Pipetters will be used to deliver 1.0 L to 2 mL volumes.6.5 Glassware:NOTE 2Sterile laboratory ware or sterile disposable laboratory wareshould be used according to standard microbiological practice.6.5.1 Glass Tubing, 6.35 mm (0.25 in.) i.d.,
34、 cut into 15 cmlengths with ends fire-polished.6.5.2 French Square Bottles, 960 mL, with metal cap.NOTE 3Alternatively, 1 L capacity canning jars may be used.6.5.3 Pipetes, Bacteriological, 10 and 2.2 mL.6.6 Incubator, capable of maintaining a temperature of 25 62C.E227503e12NOTE 4Although an incuba
35、tor is preferred, incubation may be per-formed at ambient room temperature.6.7 Manifold, aquarium style, multi-valve.NOTE 5The number of manifolds and valves per manifold willdepend on the number of microcosms in the test array. Air for eachmicrocosm shall be supplied through a single air valve. Whe
36、re used, airsterilization filters shall be placed between the air valve and microcosmaeration tube.6.8 Metal Punch, 1 cm diameter.7. Reagents and Materials7.1 Reagents:7.1.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagen
37、ts conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.67.1.2 Water PurityUnless otherwise indicated, referencesto water shall be understood to mean reagent water as definedby Type III of Specification D 1193.7.
38、1.3 Antimicrobial Pesticide(s):NOTE 6The measurement of antimicrobial pesticide (microbicide)efficacy in a medium as complex as metalworking fluid is relative, notabsolute. Consequently, when this method is used to evaluate microbicideperformance (8.3 or 8.4), it is prudent to always evaluate at lea
39、st twoantimicrobial treatments. Preferably one treatment should serve as apositive control; its efficacy in the test system having been establishedpreviously.7.1.4 Metalworking Fluid(s):NOTE 7The number of metalworking fluids available is almostlimitless. Recommendations for the use of any particula
40、r fluid cannot bemade. If the primary intent is to evaluate the general efficacy of themicrobicide(s) being tested, then it/they should be tested in various typesof formulations. If the primary intent is to protect a particular formulation,then a microbicide-free version of that formulation should b
41、e used as thecontrol and base-fluid to which the treatments are added.7.1.4.1 End-use Dilution Metalworking FluidDilute met-alworking fluid concentrate in synthetic hard water(AOAC 4.027) to achieve the concentration at which it is usedtypically in recirculating metalworking fluid systems.NOTE 8Depe
42、nding on the metalworking process, metal alloy beingworked and formulation chemistry, metalworking fluid end-use dilutionmay range from 2 % (vv)to15%(vv). If the formulation(s) being testedis (are) likely to be used at a variety of end-use strengths, they should betested minimally at the high and lo
43、w ends of the anticipated end-useconcentration range. If the test objective is to evaluate microbicideperformance in multiple metalworking fluid formulations, a 5 % (vv)end-use dilution is appropriate.7.2 Materials:7.2.1 InoculumThe microbial inoculum may vary accord-ing to the users requirements. I
44、t may be either characterized oruncharacterized. The challenge population should be accli-mated to the metalworking fluid before being used in thismethod. Acclimatization shall be achieved by growing thechallenge in the end-use dilution, negative-control metalwork-ing fluid formulation.7.2.1.1 Prepa
45、re an uncharacterized inoculum by adding 50mL of spoiled metalworking fluid to 850 mL of freshlyprepared end-use dilution, negative-control metalworkingfluid. Aerate at 25 6 2C or at ambient room temperature for24 h or until the microbial viable count reaches 109CFU mL-1. Replace 800 mL of this flui
46、d with freshly preparedportion of the negative-control fluid. Repeat the aeration andmetalworking fluid replacement procedure for a minimum ofthree cycles before using the preparation as an inoculum.7.2.1.2 Prepare a characterized inoculum by using standardmicrobiological techniques to isolate, main
47、tain and identifyspecific microbes from spoiled metalworking fluid. Alterna-tively, cultures of specific interest may be obtained from acommercial type culture collection. Examples of commercialcultures that may be used are: Aeromonas hydrophila(ATCC 13444), Candida albicans (ATCC 752), Desulfovibri
48、odesulfuricans (ATCC 7757), Escherichia coli (ATCC 8739),Flavobacterium ferrugineum (ATCC 13524), Fusarium ox-ysporum (ATCC 7601), Klebsiella pneumonia (ATCC 13883),Mycobacterium immunogenum (Rossmoore strain), Proteusmirabilis (ATCC 4675), Pseudomonas aeruginosa(ATCC 8689), Pseudomonas oleovorans (
49、ATCC 8062) andSaccharomyces cerevisiae (ATCC 2338). Before using a char-acterized inoculum for metalworking fluid bioresistance test-ing, acclimate the inoculum following the procedure describedfor an uncharacterized inoculum (7.2.1.1). Warning mi-crobes recovered from metalworking fluids may be pathogenic.Do not pipet by mouth.NOTE 9As more bioresistant metalworking fluid formulations aredeveloped, microbicide-free control fluid may not support microbialgrowth at normal end-use dilutions. If microbial viable counts do notincrease by at lea
