1、Designation: E2657 16 An American National StandardStandard Practice 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 o
2、r, 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 practice covers quantitative methods for the sam-pling and determination
3、of bacterial endotoxin concentrationsin water miscible metalworking fluids (MWF).1.2 Users of this practice need to be familiar with thehandling of MWF.1.3 This practice gives an estimate of the endotoxin con-centration in the sampled MWF.1.4 This practice replaces Method E2250.1.5 This practice see
4、ks to minimize inter-laboratory varia-tion 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 he
5、alth practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D4840 Guide for Sample Chain-of-Custody ProceduresE1488 Guide for Statistical Procedures to Use in Developingand Applying Test MethodsE1497 Practice for Selection and Saf
6、e Use of Water-Miscible and Straight Oil Metal Removal FluidsE1542 Terminology Relating to Occupational Health andSafetyE2250 Method for Determination of Endotoxin Concentra-tion in Water Miscible Metal Working Fluids (Withdrawn2008)32.2 Government Standard:429 CFR 1910.1450 Occupational Exposure to
7、 HazardousChemicals in Laboratories2.3 Other Documents:5Criteria Document for a Recommended Standard: Occupa-tional Exposure to Metalworking Fluids, 1998 NIOSHManual of Analytical Methods (NMAM), 4th ed., Eller andCassinelli, Eds., 19943. Terminology3.1 For definitions of terms relating to this prac
8、tice, refer toTerminology E1542.3.2 Definitions of Terms Specific to This Standard:3.2.1 control standard endotoxin (CSE), na purifiedpreparation of endotoxin based on the USP Reference StandardEndotoxin (RSE); used in laboratories to prepare standardsolutions.3.2.2 endotoxin, npyrogenic high molar
9、mass lipopolysac-charide (LPS) complex associated with the cell wall ofgram-negative bacteria.3.2.2.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endotoxins. Endotoxins are specifically de-tected through a Limulus Amoebocyte Lysate (LAL) test.3.2.3 endotoxin unit (EU), na biologica
10、l potency unitequivalent to the FDA Reference Standard Endotoxin (RSE).3.2.3.1 DiscussionThe current RSE (EC-6) is equivalentto 1ng = 10 EU.3.2.4 geometric mean (GM), nthe central tendency of a setof numbers expressed as the nth root of their product.3.2.5 geometric standard deviation (GSD), nthe sp
11、read ofdata in a set of numbers expressed as a geometric mean.3.2.6 Gram-negative bacteria, nprokaryotic cells thathave a complex cell wall structure that stains characteristicallywhen subjected to the differential Gram staining procedure.1This practice is under the jurisdiction of ASTM Committee E3
12、4 on Occupa-tional Health and Safety and is the direct responsibility of Subcommittee E34.50 onHealth and Safety Standards for Metal Working Fluids.Current edition approved Oct. 1, 2016. Published October 2016. Originallyapproved in 2009. Last previous edition approved in 2011 as E2657 - 11. DOI:10.
13、1520/E2657-16.2For 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.3The last approved version of this historical s
14、tandard is referenced onwww.astm.org.4Available 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.5Available from CDC/NIOSH, 4676 Columbia Pkwy, Cincinnati, OH 45226-1998.Copyright ASTM Internationa
15、l, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2.7 inhibition/enhancement phenomenon, nconditionsor artifacts in sample solutions that cause endotoxin concen-tration data from LAL assays to be less than or more than theconcentration of endotoxin actually pre
16、sent in a given aqueoussample.3.2.8 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 endotoxin.3.2.9 metalworking fluid (MWF), nany fluid used for thepurpose of cooling o
17、r treating metal surfaces during metalremoval, metal forming or surface protection or preservation.3.2.10 metal removal fluid (MRF), nany fluid in thesubclass of metalworking fluids used to cut, or otherwise takeaway material or piece of stock.3.2.10.1 DiscussionMetal removal fluids include straight
18、or neat oils (D2881), not intended 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 substancesincluding
19、, but not limited to, abrasive particles, tramp oils,cleaners, dirt, metal fines and shavings, dissolved metal andhard water salts, bacteria, fungi, microbiological decayproducts, and waste. These contaminants can cause changes inthe lubricity and cooling ability of the metal removal fluid aswell as
20、 have the potential to adversely affect the health andwelfare of employees in contact with the contaminated metalremoval fluid.3.2.11 Operator-dependent assay, nan assay performedby a technician in such a manner to cause significant influ-ence(s) on the resultant data.3.2.12 pyrogen-free (PF), adjma
21、terial(s) devoid of mea-surable endotoxin activity.3.2.13 pyrogen-free water (PFW), nprocessed water thatis devoid of measurable endotoxin activity.3.2.14 sensitivity range, na span of endotoxin measure-ments expressed as EU/mL or .4. Summary of Practice4.1 Serial dilutions of CSE in PFW in borosili
22、cate glass testtubes are prepared to construct a calibration curve.4.2 The metalworking fluid sample is sonicated,centrifuged, and the supernatant retained.4.3 Triplicates of the sample supernate, standard serialdilutions, blanks, and positive control solutions are subjected tothe kinetic chromogeni
23、c LAL assay.4.4 If data indicate interferences are present, MWF super-nate is diluted and assay is performed with diluted supernate.4.5 The reaction of Limulus amebocyte lysate with sampleendotoxin imparts a proportional yellow color to the analytesolution that is measured photometrically at 405 nm.
24、4.6 The measured endotoxin concentration is reported asEU/mL.5. Significance and Use5.1 The determination of endotoxin concentrations in MWFis a parameter that can be used in decision-making for prudentfluid management practices (fluid draining, cleaning, recharg-ing or biocide dosages).5.2 This sta
25、ndard provides a practice for analysts whoperform quantitative endotoxin analyses of water-miscibleMWF.6. 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 solution
26、s.6.2 In the event that the phenomenon of inhibition/enhancement influences this practice, endotoxin concentrationdata will be less than or more than actual concentrationspresent in a given MWF sample.6.3 LALassays are highly influenced by the skill/experiencelevel of the analyst.7. Apparatus7.1 Sam
27、pling:7.1.1 Sample Collection Container, pyrogen-free, wide-mouth, stainless steel sealable 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 rot
28、ational speed of 5000 rpm.7.2.2 Ultrasonic Water Bath, ultrasonic/water bath appara-tus with a minimum peak frequency of 40 kHz with cavitationadjustment and thermostat control; use pyrogen-free glasscontainers only.7.3 Analysis:7.3.1 Incubating/Shaking Microplate Reader, spectrophoto-metric at 405
29、nm.7.3.2 Statistical Analysis Software Package for MicroplateReader.7.3.3 Vortexer, variable speed.7.3.4 Microtiter Plates, flat-bottomed, pyrogen-free, 96-well.7.3.5 Dilution Tubes, pyrogen-free, 13 by 100 mm.7.3.6 Borosilicate Glass Test Tubes, pyrogen-free, screwcaps, 10 by 75 mm.7.3.7 Single-Cha
30、nnel Micropipetor(s), 0.5-10 L.7.3.8 Eight-Channel Micropipetor, 100 L.7.3.9 Pipet Tips, pyrogen-free, 300 L.7.3.10 Glass Rod, pyrogen-free.7.3.11 Reagent Reservoir, pyrogen-free, 8-channel multipi-pettor compatible.7.3.12 Parafilm M.E2657 1628. Reagents and Materials8.1 Control Standard Endotoxin (
31、CSE), referenced to mostcurrent Federal Drug Administration (FDA) Reference Stan-dard Endotoxin (RSE).8.2 Limulus Amebocyte Lysate (LAL), unexpired with statedpotency.8.3 Dilution Water, pyrogen-free (PFW).8.4 MWF Concentrate, concentrated, unused MWF as sup-plied.9. Hazards9.1 Aerosols of endotoxin
32、 preparations pose a potentialrespiratory hazard to susceptible laboratory personnel who aredirectly involved with an endotoxin assay.9.2 Inhalation or dermal exposure to metalworking fluidspose potential health problems for personnel involved in MWFsampling. Provision of personal protective equipme
33、nt (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 Fluids).9.3 Follow good laboratory procedures for worker protec-tion and waste disposal, including 2
34、9 CFR 1910.1450.9.4 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).10. Sampling Procedure10.1 Sampling Site:10.1.1 Select sampling site that will yield a representativeMWF sample.10.1.2 Se
35、lect individual sump(s) or central system(s) thathas actively circulating fluids. If possible, draw sample fromthe mid-point of the fluid reservoir. Otherwise, draw samplebelow the surface of the metalworking fluid volume of interestand avoid the aspiration of extraneous floating biomass.10.1.3 Use
36、aseptic techniques with pyrogen-free apparatusto aspirate a 100-mL grab sample with a glass pipet into asuitable pyrogen-free 250-mL container and then seal securelywith a pyrogen-free lid or Parafilm M.Avoid touching inner lidand interior container areas with hands/gloves or nonpyrogeniclabware.11.
37、 Sample Storage/Shipment11.1 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 bag and then refrigerate orpack in crushed ice at 4 6 2C. Avoid freezing sample, sincethis will adversely affect resu
38、ltant data.11.2 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 sample arrivesat the laboratory no later than 24 hours after its acquisition.11.3 Maintain procedures for sample custody
39、in accordancewith accepted chain of custody procedures (see Guide D4840).12. Preparation of Labware12.1 Acritical consideration of quantitative LAL analyses isthat the sample must be protected against the indiscriminateintroduction of exogenous sources of endotoxin:12.1.1 Commercially packaged labwa
40、re 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 authentication shall accom-pany labware that attests to its pyrogen-free condition. Manu-facturer ID, lot numbers, expirat
41、ion dates, and authentication/certification information shall be recorded in laboratorynotebooks.12.1.2 Commercially packaged labware that is nominallydescribed or labeled as “sterile,” “sterilized,” “disinfected,” orotherwise identified as suitable for routine microbiologicalusage only shall not be
42、 used in this standard practice, due to thepossibility of the presence of residual endotoxin on criticallabware surfaces.12.1.3 Prior to use in this standard practice, non-pyrogen-free glass or metal labware that will be used in LAL analysesshall be subjected to the depyrogenation procedure describe
43、d inSection 13 of this standard. The analyst shall not use plasticlabware, due to the possibility of introducing non-specificassay interferences, or causing container-related adsorption ofendotoxin onto surfaces, or both.13. Depyrogenation Procedure13.1 Thoroughly clean labware and then rinse twice
44、inpyrogen-free water.13.2 Bake glassware at 250C for1hinalaboratoryconvection-type oven. As part of quality assurance procedures,check oven heating performance with a NTIS-calibrated ther-mometer before each depyrogenation batch run.13.3 The analyst shall avoid indiscriminate contaminationof depyrog
45、enated labware.14. Extraction Procedure14.1 This critical procedure shall be performed by a single,experienced analyst only.14.2 Open the container with collected sample in anegative-pressure biosafety cabinet (or under a chemical fumeevacuation hood), and stir the sample vigorously with apyrogen-fr
46、ee glass rod for 1 min.14.3 Aspirate 20 mL of MWF (center, midway depth) andtransfer to a pyrogen-free test tube.14.4 Bath sonicate sample in test tube at a minimum peakfrequency of 40 kHz for1hat256 2C (or place on amechanical shaker/vortexer for 1 h).14.5 Centrifuge solution in a pyrogen-free tube
47、 at 1000 g forat least 15 min.14.6 Remove centrifuge tube and note zoning layers: trampoil (upper layer); MWF (middle layer); suspended solids(bottom layer).14.7 Pipet and discard tramp oil layer with a pyrogen-freepipet tip.E2657 16314.8 Pipet MWF layer with a pyrogen-free pipet tip.15. Microtiter
48、Plate Template Set-up15.1 Record microtiter well assignments for the 96-wellmicrotiter plate for each set of analytical solutions (in tripli-cate) in laboratory notebook.15.1.1 Samples (in triplicate).15.1.2 Standard serial solutions (in triplicate for each con-centration in the dilution series).15.
49、1.3 PFW blanks (in triplicate).15.1.4 Positive control (in triplicate record endotoxinspike concentration).NOTE 1If Parafilm M is utilized to cover vessels containing samplematerial, the extraction procedure needs to be conducted on a 1-cm2piece,and triplicates of the extract shall be subjected to LAL analysis.15.2 Program microtiter plate well locations into platereader software in accordance with predetermined templateassignments.16. Preparation of Assay Solutions16.1 Use 1.0 N HCl or 1.0 N NaOH for pH adjustment ofthe MWF sample to pH 7.5.16.2 Use fres
