1、Designation: E 2275 03 (Reapproved 2008)Standard 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 ad
2、option or, in the 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.1. Scope1.1 This practice addresses the evaluation of the relativeinherent biores
3、istance 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 fluids with a biological inoculumthat may eit
4、her 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 defined in terms of resistanceto biomass i
5、ncrease, 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 dilution. It is also applicable to metalworking
6、 fluidsthat are formulated using non-microbicidal, inherently biore-sistant components.1.4 The values stated in SI units are to be regarded as thestandard. The values given in parentheses are for informationonly.1.5 This standard does not purport to address all of thesafety concerns, if any, associa
7、ted 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:2D 888 Test Methods for Dissolved Oxygen in WaterD 1067 Test
8、Methods for Acidity or Alkalinity of WaterD 1193 Specification for Reagent WaterD 3342 Test Method for Dispersion Stability of New (Un-used) 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
9、Method for Adenosine Triphosphate (ATP)Content of Microorganisms in WaterD 4627 Test Method for Iron Chip Corrosion forWaterDilutable Metalworking FluidsD 5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating MethodsE70 Test Method for pH of Aqueous Solutions With theG
10、lass ElectrodeE 1326 Guide for Evaluating Nonconventional Microbio-logical Tests Used for 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, Pl
11、ate CountAgar49216 Direct Total Microbial Count4Microbiological Test 52.3 Government Standard: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 micr
12、o-bicidal performance.3.1.2 antimicrobial pesticide, nchemical additive regis-tered under 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 respons
13、ibility of SubcommitteeE35.15 on Antimicrobial Agents.Current edition approved April 1, 2008. Published May 2008. Originallyapproved in 2003. Last previous edition approved in 2003 as E 2275 03e1.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at
14、 serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3AOAC International Methods of Analysis, AOAC International, Gaithersburg,MD.4Available from American Public Health Association (APHA) Standard Meth-ods for the Exam
15、ination of Water and Wastewater 800 I Street, NWWashington, DC20001.5Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852-1790, http:/www.usp.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.3 as s
16、upplied, adjantimicrobial pesticide finishedproduct including the active ingredient(s), solvent and anyadditional inactive ingredients.3.1.4 biocide, nany chemical intended for use to killorganisms.3.1.5 bioresistant, adjability to withstand biological at-tack.3.1.5.1 DiscussionBioresistant, or reca
17、lcitrant, chemicalsare not readily metabolized by microorganisms.3.1.6 biostatic, adjable to prevent existing microbial con-taminants from growing or proliferating, but unable to killthem.3.1.6.1 DiscussionBiostatic additives may be registeredantimicrobial pesticides or unregistered chemicals with o
18、therperformance properties. The difference between biocidal andbiostatic performance may be attributed to dose, chemistry orboth.3.1.7 dose, nconcentration of antimicrobial pesticideadded to treated solution.3.1.7.1 DiscussionDose is generally expressed as eitherppm active ingredient (a.i.) or ppm a
19、s supplied (a.s.).3.1.8 inactive ingredient, ncomponent of antimicrobialpesticide that is not directly responsible for the pesticidesantimicrobial performance.3.1.8.1 DiscussionInactive ingredients may include, butare not limited to solvents and chemicals that improve thepesticides non-biocidal perf
20、ormance properties, such as mis-cibility and reactivity with non-target molecules in the treatedmaterial.3.1.9 minimum inhibitory concentration (MIC), nlowesttreatment-dose that will prevent test population from growing,proliferating or otherwise contributing to biodeterioration.3.2 Abbreviations:3.
21、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 more water-miscible metal-working fluids are dispensed into microcosms. The fluids maybe fresh or aged, dosed with one or more ant
22、imicrobialpesticides or undosed. Microcosms are challenged with eitheruncharacterized or characterized biological inocula. After in-oculation, microcosms are aerated either continuously or peri-odically to simulate recirculation conditions in coolant sys-tems. Chips may also be added to microcosms t
23、o simulate chipaccumulation in coolant systems.4.2 After inoculation, fluid samples are drawn from eachmicrocosm periodically and tested for the parameters ofinterest, including but not limited to microbial viable counts.Depending on the test objectives, the test duration may rangefrom 24 h to three
24、 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 to evaluate metalworkingfluid formulation resistance to repeated challenges. For testslasting longer than one-week, 10 to 80 % of th
25、e fluid isexchanged weekly with fresh fluid before the additionalchallenge. The percentage of fluid exchange should reflectanticipated fluid turnover rates in fluids end-use application.4.3 Bioresistance is determined as the test fluids relativeability to prevent the proliferation of challenge micro
26、bes, retainits original chemical or physical properties of some combina-tion of the above. The bioresistance of test formulations isdefined relative to that of a benchmark or control formulation.5. Significance and Use5.1 This practice provides laboratory procedures for ratingthe relative bioresista
27、nce of metalworking fluid formulations,for determining the need for microbicide addition prior to orduring fluid use in metalworking systems and for evaluatingmicrobicide performance. General considerations for microbi-cide selection are provided in Practice E 2169.5.2 The factors affecting challeng
28、e population numbers,taxonomic diversity, physiological state, inoculation frequencyand biodeterioration effects in recirculating metalworking fluidsystems are varied and only partially understood. Conse-quently, the results of tests completed in accordance with thispractice should be used only to c
29、ompare the relative perfor-mance of products or microbicide treatments included in a testseries. Results should not be construed as predicting actualfield performance.6. Apparatus6.1 Air Supply, air provided at no more than 110 kPa.NOTE 1Any air source that is free of organic vapors, organic mattero
30、r other objectionable material may be used. Sterile air need not be usedfor the uncharacterized inoculum, but shall be used for the characterizedinoculum. If necessary, air may be sterilized either by inserting, in series,two commercially available in-line sterile filters designed for this purpose.A
31、lternatively 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 stoppers, glass tubing and neoprene tubing. Wraploosely in aluminum foil and steam sterilize at 103 to 138 kPa (15 to 20psi) for 30 min
32、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 filter, average lifetime incontinuous use is two weeks. Discard sooner if upstream filter becomeswet or contaminated with oil.6.2 Aq
33、uarium 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 Adjustable Volume Pipetters, with sterile disposabletips. Pipetters will be used to deliver 1.0 L to 2 mL volumes.6.5 Glassware:NOTE
34、 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., cut into 15 cmlengths with ends fire-polished.6.5.2 French Square Bottles, 960 mL, with metal cap.NOTE 3Alternatively, 1 L capacit
35、y 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.E 2275 03 (2008)2NOTE 4Although an incubator is preferred, incubation may be per-formed at ambient room temperature.6.7 Manifold, aquarium style, multi-valve.NOTE 5T
36、he 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. Where used, airsterilization filters shall be placed between the air valve and microcosmaeration tube.6.8 Metal Punch, 1 cm dia
37、meter.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 reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifica
38、tions 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 1193.7.1.3 Antimicrobial Pesticide(s):NOTE 6The measurement of antimicrobial pesticide (microbicide)efficacy in a medium as complex
39、 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 least twoantimicrobial treatments. Preferably one treatment should serve as apositive control; its efficacy in the test system
40、having been establishedpreviously.7.1.4 Metalworking Fluid(s):NOTE 7The number of metalworking fluids available is almostlimitless. Recommendations for the use of any particular fluid cannot bemade. If the primary intent is to evaluate the general efficacy of themicrobicide(s) being tested, then it/
41、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 be used as thecontrol and base-fluid to which the treatments are added.7.1.4.1 End-use Dilution Metalworking FluidDilute met-
42、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 8Depending on the metalworking process, metal alloy beingworked and formulation chemistry, metalworking fluid end-use dilutionmay
43、 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 low ends of the anticipated end-useconcentration range. If the test objective is to evaluate microbicideperformance in multipl
44、e 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. It may be either characterized oruncharacterized. The challenge population should be accli-mated to the metalworking fluid be
45、fore 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 Prepare an uncharacterized inoculum by adding 50mL of spoiled metalworking fluid to 850 mL of freshlyprepared end-use dilution, n
46、egative-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 fluid with freshly preparedportion of the negative-control fluid. Repeat the aeration andmetalworking fluid replacement procedur
47、e 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, maintain and identifyspecific microbes from spoiled metalworking fluid. Alterna-tively, cultures of specific interest may be obt
48、ained from acommercial type culture collection. Examples of commercialcultures that may be used are: Aeromonas hydrophila(ATCC 13444), Candida albicans (ATCC 752), Desulfovibriodesulfuricans (ATCC 7757), Escherichia coli (ATCC 8739),Flavobacterium ferrugineum (ATCC 13524), Fusarium ox-ysporum (ATCC
49、7601), Klebsiella pneumonia (ATCC 13883),Mycobacterium immunogenum (Rossmoore strain), Proteusmirabilis (ATCC 4675), Pseudomonas aeruginosa(ATCC 8689), Pseudomonas oleovorans (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). WarningMicrobes recovered from metalworking fluids may be patho-genic. Do not pipet by mouth.NOTE 9As more bioresistant metalwork
