1、Designation: E 2657 09An American National StandardStandard Test Method forDetermination of Endotoxin Concentrations in Water-Miscible Metalworking Fluids1This standard is issued under the fixed designation E 2657; the number immediately following the designation indicates the year oforiginal adopti
2、on or, in the case of revision, the year of last revision. 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 test method covers quantitative methods for thesampling and determina
3、tion of bacterial endotoxin concentra-tions in water miscible metalworking fluids (MWF).1.2 Users of this test method need to be familiar with thehandling of MWF.1.3 This test method gives an estimate of the endotoxinconcentration in the sampled MWF.1.4 This test method replaces E 2250.1.5 This test
4、 method seeks to minimize inter-laboratoryvariation of endotoxin data but does not ensure uniformity ofresults.1.6 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 appro-priate safe
5、ty and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 2881 Classification for Metal Working Fluids and RelatedMaterialsD 4840 Guide for Sample Chain-of-Custody ProceduresE 1497 Practice for Selection and Safe Use
6、of Water-Miscible and Straight Oil Metal Removal FluidsE 1542 Terminology Relating to Occupational Health andSafety2.2 Government Standard:329 CFR 1910.1450 Occupational Exposure to HazardousChemicals in Laboratories29 CFR 1910.1000 Air Contaminants2.3 Other Documents:Criteria Document for a Recomme
7、nded Standard: Occupa-tional Exposure to Metalworking Fluids, 1998NIOSH Manual of Analytical Methods (NMAM), 4th ed.,Eller and Cassinelli, Eds., 19943. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 control standard endotoxin (CSE), na purifiedpreparation of endotoxin based on t
8、he USP Reference StandardEndotoxin (RSE); used in laboratories to prepare standardsolutions.3.1.2 endotoxin, na lipopolysaccharide derived from theouter membrane of Gram-negative bacteria.3.1.3 endotoxin unit (EU), na biological potency unitequivalent to the FDA Reference Standard Endotoxin (RSE).3.
9、1.3.1 DiscussionThe current RSE (EC-6) is equivalentto 1ng = 10 EU.3.1.4 geometric mean (GM), nthe central tendency of aset of numbers expressed as the nth root of their product.3.1.5 geometric standard deviation (GSD), nthe spread ofdata in a set of numbers expressed as a geometric mean.3.1.6 gram-
10、negative bacteria, nprokaryotic cells thathave a complex cell wall structure that stains characteristicallywhen subjected to the differential Gram staining procedure.3.1.7 inhibition/enhancement phenomenon, nconditionsor artifacts in sample solutions that cause endotoxin concen-tration data from LAL
11、 assays to be less than or more than theconcentration of endotoxin actually present in a given aqueoussample.3.1.8 Limulus amebocyte lysate (LAL) assay, na biologi-cal assay dependent on a series of cascading enzyme reactionsthat occur when Limulus blood cell (amebocyte) lysate com-bines with endoto
12、xin.3.1.9 metalworking fluid (MWF), nany fluid used for thepurpose of cooling or treating metal surfaces during metalremoval, metal forming or surface protection or preservation.3.1.10 metal removal fluid (MRF), nany fluid in thesubclass of metalworking fluids used to cut, or otherwise takeaway mate
13、rial or piece of stock.1This test method is under the jurisdiction of ASTM Committee E34 onOccupational Health and Safety and is the direct responsibility of SubcommitteeE34.50 on Health and Safety Standards for Metal Working Fluids.Current edition approved May 1, 2009. Published June 2009.2For refe
14、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.3Available from U.S. Government Printing Office Superintendent of Docu
15、ments,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.10.1 DiscussionMetal removal fluids include straightor neat oils (see D 2881), not inten
16、ded for further dilution withwater, and water miscible soluble oils, semisynthetics andsynthetics, which are intended to be diluted with water beforeuse. Metal removal fluids become contaminated during use inthe workplace with a variety of workplace substances includ-ing, but not limited to, abrasiv
17、e particles, tramp oils, cleaners,dirt, metal fines and shavings, dissolved metal and hard watersalts, bacteria, fungi, microbiological decay products, andwaste. These contaminants can cause changes in the lubricityand cooling ability of the metal removal fluid as well as havethe potential to advers
18、ely affect the health and welfare ofemployees in contact with the contaminated metal removalfluid.3.1.11 operator-dependent assay, nan assay performed bya technician in such a manner to cause significant influence(s)on the resultant data.3.1.12 pyrogen-free (PF), adjmaterial(s) devoid of mea-surable
19、 endotoxin activity.3.1.13 pyrogen-free water (PFW), nprocessed water thatis devoid of measurable endotoxin activity.3.1.14 sensitivity range, na span of endotoxin measure-ments expressed as EU/mL or l.4. Summary of Test Method4.1 Serial dilutions of CSE in PFW in borosilicate glass testtubes are pr
20、epared to construct a calibration curve.4.2 The metalworking fluid sample is sonicated, centri-fuged, and the supernatant retained.4.3 Triplicates of the sample supernatant, standard serialdilutions, blanks, and positive control solutions are subjected tothe kinetic chromogenic LAL assay.4.4 The rea
21、ction of Limulus amebocyte lysate with sampleendotoxin imparts a proportional yellow color to the analytesolution that is measured photometrically at 405 nm.4.5 The measured endotoxin concentration is reported asEU/mL.5. Significance and Use5.1 The determination of endotoxin concentrations in met-al
22、working fluids is a parameter that can be used in decision-making for prudent fluid management practices (fluid draining,cleaning, recharging or biocide dosages).5.2 This test method provides a test method for analystswho perform quantitative endotoxin analyses of water-misciblemetalworking fluids.6
23、. Interferences6.1 Data from samples analyzed by LAL methodologies areprone to variations due to batch differences in lysatecomposition/processing, non-optimal pH and temperatures ofassay solutions.6.2 In the event that the phenomenon of inhibition/enhancement influences this test method, endotoxin
24、concentra-tion data will be less than or more than actual concentrationspresent in a given metalworking fluid sample.6.3 LALassays are highly influenced by the skill/experiencelevel of the analyst.7. Apparatus7.1 Sampling:7.1.1 Sample Collection Container, pyrogen-free, wide-mouth, stainless steel s
25、ealable container, at least 100 mLcapacity.7.1.2 Glass Pipet, pyrogen-free, 50 mL.7.1.3 Battery-Powered Aspirator Unit (or Suction Bulb),compatible with 100 mL glass pipet.7.2 Extraction:7.2.1 Centrifuge, minimum rotational speed of 5000 rpm.7.2.2 Ultrasonic Water Bath, ultrasonic/water bath appara-
26、tus with a minimum peak frequency of 40 kHz with cavitationadjustment and thermostat control; use pyrogen-free glasscontainers only.7.3 Reagents and Materials:7.3.1 Control Standard Endotoxin (CSE), referenced tomost current Federal Drug Administration (FDA) ReferenceStandard Endotoxin (RSE).7.3.2 L
27、imulus Amebocyte Lysate (LAL), unexpired withstated potency.7.3.3 Dilution water, pyrogen-free.7.4 Analysis:7.4.1 Incubating/Shaking Microplate Reader, spectrophoto-metric at 405 nm.7.4.2 Statistical Analysis Software Package for MicroplateReader7.4.3 Vortexer, variable speed.7.4.4 Microtiter Plates
28、, flat-bottomed, pyrogen-free, 96-well.7.4.5 Dilution Tubes, pyrogen-free, 13 by 100 mm.7.4.6 Borosilicate Glass Test Tubes, pyrogen-free, screwcaps, 10 by 75 mm.7.4.7 Single-Channel Micropipettor(s), 0.5-10 L.7.4.8 Eight-Channel Micropipettor, 100 L.7.4.9 Pipet Tips, pyrogen-free, 300 L.7.4.10 Glas
29、s Rod, pyrogen-free.7.4.11 Reagent Reservoir, pyrogen-free, 8-channel multipi-pettor compatible.7.4.12 Parafilm M8. Hazards8.1 Aerosols of endotoxin preparations pose a potentialrespiratory hazard to susceptible laboratory personnel who aredirectly involved with an endotoxin assay.8.2 Inhalation or
30、dermal exposure to metalworking fluidspose potential health problems for personnel involved in MWFsampling. Provision of personal protective equipment (PPE) inthe form of respirators or protective clothing, or both, ispotentially indicated (see Practice E 1497 and Criteria Docu-ment for a Recommende
31、d Standard: Occupational Exposure toMetalworking Fluids).8.3 Follow good laboratory procedures for worker protec-tion and waste disposal, including 29 CFR 1910.1450.8.4 Review material safety data sheets (MSDS) for materi-als in use at a facility to identify potential hazards to determineappropriate
32、 PPE (see 29 CFR 1910.1000).9. Sampling9.1 Sampling Site:E26570929.1.1 Select sampling site that will yield a representativemetalworking fluid sample.9.1.2 Select individual sump(s) or central system(s) that hasactively circulating fluids. If possible, draw sample from themid-point of the fluid rese
33、rvoir. Otherwise, draw sample belowthe surface of the metalworking fluid volume of interest andavoid the aspiration of extraneous floating biomass.9.1.3 Use aseptic techniques with pyrogen-free apparatus toaspirate a 100 mL grab sample with a glass pipet into a suitablepyrogen-free 250 mL container
34、and then seal securely with apyrogen-free lid or Parafilm M. Avoid touching inner lid andinterior container areas with hands/gloves or nonpyrogeniclabware.10. Sample Storage/Shipment10.1 For best results, LAL analysis of the sample within 24hours is advisable. However, if this is not feasible, store
35、 thesealed sample container in a plastic bag and then refrigerate orpack in crushed ice at 4 6 2C. Avoid freezing sample, sincethis will adversely affect resultant data.10.2 If the sample is shipped to an analytical laboratory,pack its container securely in cold packs (or portable refrig-eration) an
36、d expedite shipment time so that the sample arrivesat the laboratory no later than 24 hours after its acquisition.10.3 Maintain procedures for sample custody in accordancewith accepted chain of custody procedures (see Guide D 4840).11. Preparation of Labware11.1 Acritical consideration of quantitati
37、ve LAL analyses isthat the sample must be protected against the indiscriminateintroduction of exogenous sources of endotoxin:11.1.1 Commercially-packaged labware used in LAL analy-ses shall be clearly marked as pyrogen-free, endotoxin-free,depyrogenated, or clearly identified as suitable for use in
38、LALanalyses. A certificate of authentication shall accompany lab-ware that attests to its pyrogen-free condition. ManufacturerID, lot numbers, expiration dates, and authentication/certification information shall be recorded in laboratory note-books.11.1.2 Commercially-packaged labware that is nomina
39、llydescribed or labeled as sterile, sterilized, disinfected, or other-wise identified as suitable for routine microbiological usageonly shall not be used in this test method, due to the possibilityof the presence of residual endotoxin on critical labwaresurfaces.11.1.3 Prior to use in this test meth
40、od, non-pyrogen-freeglass or metal labware that will be used in LAL analyses shallbe subjected to the depyrogenation procedure described inSection 12. The analyst shall not use plastic labware, due to thepossibility of introducing non-specific assay interferencesand/or causing container-related adso
41、rption of endotoxin ontosurfaces.12. Depyrogenation Procedure12.1 Thoroughly clean labware and then rinse twice inpyrogen-free water.12.2 Bake glassware at 250C for1hinalaboratoryconvection-type oven. As part of quality assurance procedures,check oven heating performance with a NTIS-calibrated ther-
42、mometer before each depyrogenation batch run.12.3 The analyst shall avoid indiscriminate contaminationof depyrogenated labware.13. Extraction Procedure13.1 This critical procedure shall be performed by a single,experienced analyst only.13.2 Open the container with collected sample in anegative-press
43、ure biosafety cabinet (or under a chemical fumeevacuation hood), and stir the sample vigorously with apyrogen-free glass rod for 1 min.13.3 Aspirate 20 mL of metalworking fluid (center, midwaydepth) and transfer to a pyrogen-free test tube.13.4 Bath sonicate sample in test tube at a minimum peakfreq
44、uency of 40 kHz for1hat256 2C (or place on amechanical shaker/vortexer for 1 h).13.5 Centrifuge solution in a pyrogen-free tube at 1000 g forat least 15 min.13.6 Remove centrifuge tube and note zoning layers: trampoil (upper layer); metalworking fluid (middle layer); suspendedsolids (bottom layer).1
45、3.7 Pipet and discard tramp oil layer with a pyrogen-freepipet tip.13.8 Pipet metalworking fluid layer with a pyrogen-freepipet tip.14. Microtiter Plate Template Set-up14.1 Record microtiter well assignments for the 96-wellmicrotiter plate for each set of analytical solutions (in tripli-cate) in lab
46、oratory notebook.14.1.1 Samples (in triplicate).14.1.2 Standard serial solutions (in triplicate for each con-centration in the dilution series).14.1.3 PFW blanks (in triplicate).14.1.4 Positive control (in triplicaterecord endotoxinspike concentration).NOTE 1If Parafilm M is utilized to cover vessel
47、s containing samplematerial, the extraction procedure needs to be conducted on a 1-cm2piece,and triplicates of the extractate shall be subjected to LAL analysis.14.2 Program microtiter plate well locations into platereader software in accordance with predetermined templateassignments.15. Preparation
48、 of Assay Solutions15.1 Use 1.0 N HCl or 1.0 N NaOH for pH adjustment ofthe MWF sample to pH 7.5.15.2 Use fresh (non-expired) PFW for use as a diluent forlyophilized Control Standard Endotoxin (CSE), Limulus ame-bocyte lysate (LAL), blanks, and positive controls. PFW fromthe same lot shall be used f
49、or all analyte solutions per assay.15.3 Reconstitute CSE in accordance with manufacturersdirections and adjust to pH 7.5.15.4 Standard Solutions:15.4.1 Use CSE solution to prepare serial dilutions withconcentrations of 5.0, 0.5, 0.05, and 0.005 EU/mL.15.4.2 Dispense 100 L of each CSE dilution (in triplicate)in designated LAL reagent grade microtiter plate wells.E265709315.5 Blanks:15.5.1 Dispense 100 L of 7.5 pH-adjusted PFW (in tripli-cate) in designated microtiter plate wells.15.6 Positive Controls:15.6.1 Dispense 100 L of extract solution (in triplicate
