1、Designation: E2657 11Standard Test Method forDetermination of Endotoxin Concentrations in Water-Miscible Metalworking Fluids1This standard is issued under the fixed designation E2657; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision,
2、 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 determination of bacterial endotoxin con
3、centra-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 method gives an estimate of the endotoxin concen-tration in the sampled MWF.1.4 This method replaces Test Method E2250.1.5 This test method seeks to minimize in
4、ter-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 safety and health practices and
5、determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D4840 Guide for Sample Chain-of-Custody ProceduresE1497 Practice for Selection and Safe Use of Water-Miscible and Straight Oil Metal Removal FluidsE1542 Terminology Relating to Occupational
6、 Health andSafety2.2 Government Standard:329 CFR 1910.1450 Occupational Exposure to HazardousChemicals in Laboratories2.3 Other Documents:4Criteria Document for a Recommended Standard: Occupa-tional Exposure to Metalworking Fluids, 1998 NIOSHManual of Analytical Methods (NMAM), 4th ed., Eller andCas
7、sinelli, Eds., 19943. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 control standard endotoxin (CSE), na purifiedpreparation of endotoxin based on the USP Reference StandardEndotoxin (RSE); used in laboratories to prepare standardsolutions.3.1.2 endotoxin, npyrogenic high molar
8、 mass lipopoly-saccharide (LPS) complex associated with the cell wall ofgram-negative bacteria.3.1.2.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endotoxins. Endotoxins are specifically de-tected through a Limulus Amoebocyte Lysate (LAL) test.3.1.3 endotoxin unit (EU), na biologic
9、al potency unitequivalent to the FDA Reference Standard Endotoxin (RSE).3.1.3.1 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 s
10、pread ofdata in a set of numbers expressed as a geometric mean.3.1.6 Gram-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 artif
11、acts in sample solutions that cause endotoxin concen-tration data from LAL 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 reactionst
12、hat occur when Limulus blood cell (amebocyte) lysate com-bines with endotoxin.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
13、thesubclass of metalworking fluids used to cut, or otherwise takeaway material 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
14、Fluids.Current edition approved Dec. 1, 2011. Published January 2012. Originallyapproved in 2009. Last previous edition approved in 2009 as E2657 - 09. DOI:10.1520/E2657-11.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For A
15、nnual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.4Available from CDC/NIOSH
16、, 4676 Columbia Pkwy, Cincinnati, OH 45226-1998.1Copyright 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 (D2881), not intended for further dilution withwater, and water misc
17、ible 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, abrasive particles, tramp oils, cleaners,dirt, metal fine
18、s 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 adversely affect the health and welfare ofemployees in c
19、ontact with the contaminated metal removalfluid.3.1.11 Operator-dependent assay, nan assay performedby a technician in such a manner to cause significant influ-ence(s) on the resultant data.3.1.12 pyrogen-free (PF), adjmaterial(s) devoid of mea-surable endotoxin activity.3.1.13 pyrogen-free water (P
20、FW), 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 prepared to construct a calibration curve.4.2 The
21、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 reaction of Limulus amebocyte lysate with sampleend
22、otoxin 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-alworking fluids is a parameter that can be used i
23、n decision-making for prudent fluid management practices (fluid draining,cleaning, recharging or biocide dosages).5.2 This standard provides a test method for analysts whoperform quantitative endotoxin analyses of water-misciblemetalworking fluids.6. Interferences6.1 Data from samples analyzed by LA
24、L 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 concentra-tion data will be less than or more than
25、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 sealable container, at least 100 mLcapacity.7.1.2 Gl
26、ass 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-tus with a minimum peak frequency of 40 kHz with ca
27、vitationadjustment 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 Limulus Amebocyte Lysate (LAL), unexpired withstated
28、 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 MicroplateReader.7.4.3 Vortexer, variable speed.7.4.4 Microtiter Plates, flat-bottomed, pyrogen-free, 96-well.7.4.5 Dilut
29、ion 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 Micropipetor(s), 0.5-10 L.7.4.8 Eight-Channel Micropipetor, 100 L.7.4.9 Pipet Tips, pyrogen-free, 300 L.7.4.10 Glass Rod, pyrogen-free.7.4.11 Reagent Reservoir, pyroge
30、n-free, 8-channel multipi-pettor compatible.7.4.12 Parafilm M.8. 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 dermal exposure to metalworking fluidspose potentia
31、l 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 E1497 and Criteria Docu-ment for a Recommended Standard: Occupational Exposure toMetalworking Flu
32、ids).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 PPE (see 29 CFR 1910.1000).9. Sampling Procedure9.1
33、 Sampling Site:E2657 1129.1.1 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 reservoir. Otherwise, draw sample belowthe su
34、rface 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 and then seal securely with apyrogen-free
35、 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 thesealed sample container in a plastic
36、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) and expedite shipment time so that the samp
37、le 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 D4840).11. Preparation of Labware11.1 Acritical consideration of quantitative LAL analyses isthat the sample must be
38、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 usein LAL analyses. A certificate of authe
39、ntication shall accom-pany labware that attests to its pyrogen-free condition. Manu-facturer ID, lot numbers, expiration dates, and authentication/certification information shall be recorded in laboratorynotebooks.11.1.2 Commercially packaged labware that is nominallydescribed or labeled as “sterile
40、,” “sterilized,” “disinfected,” orotherwise identified as suitable for routine microbiologicalusage only shall not be used in this standard practice, due to thepossibility of the presence of residual endotoxin on criticallabware surfaces.11.1.3 Prior to use in this standard practice, non-pyrogen-fre
41、e glass or metal labware that will be used in LAL analysesshall be subjected to the depyrogenation procedure described inSection 12 of this standard. The analyst shall not use plasticlabware, due to the possibility of introducing non-specificassay interferences, or causing container-related adsorpti
42、on ofendotoxin onto surfaces, or both.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
43、ther-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-
44、pressure 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 pea
45、kfrequency 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 lay
46、er).13.7 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) i
47、n laboratory 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 triplicate record endotoxinspike concentration).NOTE 1If Parafilm M is utilized to cover
48、vessels 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. Prepa
49、ration 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 for all analyte solutions per assay.15.3 Reconstitute CSE as per manufacturers directions andadjust 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 10
