1、Designation: E2657 11E2657 16 An American National StandardStandard Test Method 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 o
2、foriginal adoption 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 the sampl
3、ing and determination of bacterial endotoxin concentrations inwater miscible metalworking fluids (MWF).1.2 Users of this test method need to be familiar with the handling of MWF.1.3 This method gives an estimate of the endotoxin concentration in the sampled MWF.1.4 This method replaces Test Method E
4、2250.1.5 This test method seeks to minimize inter-laboratory variation of endotoxin data but does not ensure uniformity of results.1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establ
5、ish appropriate safety and health practices and determine the applicability of regulatorylimitations 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 Fl
6、uidsE1542 Terminology Relating to Occupational Health and Safety2.2 Government Standard:329 CFR 1910.1450 Occupational Exposure to Hazardous Chemicals in Laboratories2.3 Other Documents:4Criteria Document for a Recommended Standard: Occupational Exposure to Metalworking Fluids, 1998 NIOSHManual of A
7、nalytical 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 purified preparation of endotoxin based on the USPReference Standard Endotoxin(RSE); used in laboratories to prepare standard
8、 solutions.3.1.2 endotoxin, npyrogenic high molar mass lipopolysaccharide (LPS) complex associated with the cell wall ofgram-negative bacteria.3.1.2.1 Discussion1 This test method practice is under the jurisdiction of ASTM Committee E34 on Occupational Health and Safety and is the direct responsibil
9、ity of Subcommittee E34.50on Health and Safety Standards for Metal Working Fluids.Current edition approved Dec. 1, 2011Oct. 1, 2016. Published January 2012October 2016. Originally approved in 2009. Last previous edition approved in 20092011 asE2657 - 09.E2657 - 11. DOI: 10.1520/E2657-11.10.1520/E265
10、7-16.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.4 Available from U.S. Government Printing Office Superin
11、tendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.5 Available from CDC/NIOSH, 4676 Columbia Pkwy, Cincinnati, OH 45226-1998.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of w
12、hat changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered t
13、he official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Though endotoxins are pyrogens, not all pyrogens are endotoxins. Endotoxins are specifically detected through a LimulusAmoebocyte Lysate (LAL) test.3.1.3 endotoxin u
14、nit (EU), na biological potency unit equivalent to the FDA Reference Standard Endotoxin (RSE).3.1.3.1 DiscussionThe current RSE (EC-6) is equivalent to 1ng = 10 EU.3.1.4 geometric mean (GM), nthe central tendency of a set of numbers expressed as the nth root of their product.3.1.5 geometric standard
15、 deviation (GSD), nthe spread of data in a set of numbers expressed as a geometric mean.3.1.6 Gram-negative bacteria, nprokaryotic cells that have a complex cell wall structure that stains characteristically whensubjected to the differential Gram staining procedure.3.1.7 inhibition/enhancement pheno
16、menon, nconditions or artifacts in sample solutions that cause endotoxin concentrationdata from LAL assays to be less than or more than the concentration of endotoxin actually present in a given aqueous sample.3.1.8 Limulus amebocyte lysate (LAL) assay, na biological assay dependent on a series of c
17、ascading enzyme reactions thatoccur when Limulus blood cell (amebocyte) lysate combines with endotoxin.3.1.9 metalworking fluid (MWF), nany fluid used for the purpose of cooling or treating metal surfaces during metal removal,metal forming or surface protection or preservation.3.1.10 metal removal f
18、luid (MRF), nany fluid in the subclass of metalworking fluids used to cut, or otherwise take awaymaterial or piece of stock.3.1.10.1 DiscussionMetal removal fluids include straight or neat oils (D2881), not intended for further dilution with water, and water miscible solubleoils, semisynthetics and
19、synthetics, which are intended to be diluted with water before use. Metal removal fluids becomecontaminated during use in the workplace with a variety of workplace substances including, but not limited to, abrasive particles,tramp oils, cleaners, dirt, metal fines and shavings, dissolved metal and h
20、ard water salts, bacteria, fungi, microbiological decayproducts, and waste. These contaminants can cause changes in the lubricity and cooling ability of the metal removal fluid as wellas have the potential to adversely affect the health and welfare of employees in contact with the contaminated metal
21、 removal fluid.3.1.11 Operator-dependent assay, nan assay performed by a technician in such a manner to cause significant influence(s) onthe resultant data.3.1.12 pyrogen-free (PF), adjmaterial(s) devoid of measurable endotoxin activity.3.1.13 pyrogen-free water (PFW), nprocessed water that is devoi
22、d of measurable endotoxin activity.3.1.14 sensitivity range, na span of endotoxin measurements expressed as EU/mL or .4. Summary of Test Method4.1 Serial dilutions of CSE in PFW in borosilicate glass test tubes are prepared to construct a calibration curve.4.2 The metalworking fluid sample is sonica
23、ted, centrifuged, and the supernatant retained.4.3 Triplicates of the sample supernatant, standard serial dilutions, blanks, and positive control solutions are subjected to thekinetic chromogenic LAL assay.4.4 The reaction of Limulus amebocyte lysate with sample endotoxin imparts a proportional yell
24、ow color to the analyte solutionthat is measured photometrically at 405 nm.4.5 The measured endotoxin concentration is reported as EU/mL.5. Significance and Use5.1 The determination of endotoxin concentrations in metalworking fluids is a parameter that can be used in decision-makingfor prudent fluid
25、 management practices (fluid draining, cleaning, recharging or biocide dosages).5.2 This standard provides a test method for analysts who perform quantitative endotoxin analyses of water-misciblemetalworking fluids.6. Interferences6.1 Data from samples analyzed by LAL methodologies are prone to vari
26、ations due to batch differences in lysatecomposition/processing, non-optimal pH and temperatures of assay solutions.E2657 1626.2 In the event that the phenomenon of inhibition/enhancement influences this test method, endotoxin concentration data willbe less than or more than actual concentrations pr
27、esent in a given metalworking fluid sample.6.3 LAL assays are highly influenced by the skill/experience level of the analyst.7. Apparatus7.1 Sampling:7.1.1 Sample Collection Container, pyrogen-free, wide-mouth, stainless steel sealable container, at least 100 mL capacity.7.1.2 Glass Pipet, pyrogen-f
28、ree, 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 apparatus with a minimum peak frequency of 40 kHz with cavitationadjustment a
29、nd thermostat control; use pyrogen-free glass containers only.7.3 Reagents and Materials:7.3.1 Control Standard Endotoxin (CSE), referenced to most current Federal Drug Administration (FDA) Reference StandardEndotoxin (RSE).7.3.2 Limulus Amebocyte Lysate (LAL), unexpired with stated potency.7.3.3 Di
30、lution Water, pyrogen-free.7.4 Analysis:7.4.1 Incubating/Shaking Microplate Reader, spectrophotometric at 405 nm.7.4.2 Statistical Analysis Software Package for Microplate Reader.7.4.3 Vortexer, variable speed.7.4.4 Microtiter Plates, flat-bottomed, pyrogen-free, 96-well.7.4.5 Dilution Tubes, pyroge
31、n-free, 13 by 100 mm.7.4.6 Borosilicate Glass Test Tubes, pyrogen-free, screw caps, 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, pyrogen-free, 8-channe
32、l multipipettor compatible.7.4.12 Parafilm M.8. Hazards8.1 Aerosols of endotoxin preparations pose a potential respiratory hazard to susceptible laboratory personnel who are directlyinvolved with an endotoxin assay.8.2 Inhalation or dermal exposure to metalworking fluids pose potential health proble
33、ms for personnel involved in MWFsampling. Provision of personal protective equipment (PPE) in the form of respirators or protective clothing, or both, is potentiallyindicated (see Practice E1497 and Criteria Document for a Recommended Standard: Occupational Exposure to MetalworkingFluids).8.3 Follow
34、 good laboratory procedures for worker protection and waste disposal, including 29 CFR 1910.1450.8.4 Review material safety data sheets (MSDS) for materials in use at a facility to identify potential hazards to determineappropriate PPE (see 29 CFR 1910.1000).9. Sampling Procedure9.1 Sampling Site:9.
35、1.1 Select sampling site that will yield a representative metalworking fluid sample.9.1.2 Select individual sump(s) or central system(s) that has actively circulating fluids. If possible, draw sample from themid-point of the fluid reservoir. Otherwise, draw sample below the surface of the metalworki
36、ng fluid volume of interest and avoidthe aspiration of extraneous floating biomass.9.1.3 Use aseptic techniques with pyrogen-free apparatus to aspirate a 100-mL grab sample with a glass pipet into a suitablepyrogen-free 250-mL container and then seal securely with a pyrogen-free lid or Parafilm M.Av
37、oid touching inner lid and interiorcontainer areas with hands/gloves or nonpyrogenic labware.E2657 16310. Sample Storage/Shipment10.1 For best results, LAL analysis of the sample within 24 hours is advisable. However, if this is not feasible, store the sealedsample container in a plastic bag and the
38、n refrigerate or pack in crushed ice at 4 6 2C. Avoid freezing sample, since this willadversely affect resultant data.10.2 If the sample is shipped to an analytical laboratory, pack its container securely in cold packs (or portable refrigeration) andexpedite shipment time so that the sample arrives
39、at the laboratory no later than 24 hours after its acquisition.10.3 Maintain procedures for sample custody in accordance with accepted chain of custody procedures (see Guide D4840).11. Preparation of Labware11.1 A critical consideration of quantitative LAL analyses is that the sample must be protect
40、ed against the indiscriminateintroduction of exogenous sources of endotoxin:11.1.1 Commercially packaged labware used in LAL analyses shall be clearly marked as “pyrogen-free,” “endotoxin-free,”“depyrogenated,” or clearly identified as suitable for use in LAL analyses.Acertificate of authentication
41、shall accompany labwarethat attests to its pyrogen-free condition. Manufacturer ID, lot numbers, expiration dates, and authentication/certificationinformation shall be recorded in laboratory notebooks.11.1.2 Commercially packaged labware that is nominally described or labeled as “sterile,” “steriliz
42、ed,” “disinfected,” orotherwise identified as suitable for routine microbiological usage only shall not be used in this standard practice, due to thepossibility of the presence of residual endotoxin on critical labware surfaces.11.1.3 Prior to use in this standard practice, non-pyrogen-free glass or
43、 metal labware that will be used in LAL analyses shallbe subjected to the depyrogenation procedure described in Section 12 of this standard. The analyst shall not use plastic labware,due to the possibility of introducing non-specific assay interferences, or causing container-related adsorption of en
44、dotoxin ontosurfaces, or both.12. Depyrogenation Procedure12.1 Thoroughly clean labware and then rinse twice in pyrogen-free water.12.2 Bake glassware at 250C for 1 h in a laboratory convection-type oven.As part of quality assurance procedures, check ovenheating performance with a NTIS-calibrated th
45、ermometer before each depyrogenation batch run.12.3 The analyst shall avoid indiscriminate contamination of 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 a negative-
46、pressure biosafety cabinet (or under a chemical fume evacuationhood), and stir the sample vigorously with a pyrogen-free glass rod for 1 min.13.3 Aspirate 20 mL of metalworking fluid (center, midway depth) and transfer to a pyrogen-free test tube.13.4 Bath sonicate sample in test tube at a minimum p
47、eak frequency of 40 kHz for 1 h at 25 6 2C (or place on a mechanicalshaker/vortexer for 1 h).13.5 Centrifuge solution in a pyrogen-free tube at 1000 g for at least 15 min.13.6 Remove centrifuge tube and note zoning layers: tramp oil (upper layer); metalworking fluid (middle layer); suspendedsolids (
48、bottom layer).13.7 Pipet and discard tramp oil layer with a pyrogen-free pipet tip.13.8 Pipet metalworking fluid layer with a pyrogen-free pipet tip.14. Microtiter Plate Template Set-up14.1 Record microtiter well assignments for the 96-well microtiter plate for each set of analytical solutions (in t
49、riplicate) inlaboratory notebook.14.1.1 Samples (in triplicate).14.1.2 Standard serial solutions (in triplicate for each concentration in the dilution series).14.1.3 PFW blanks (in triplicate).14.1.4 Positive control (in triplicate record endotoxin spike concentration).NOTE 1If Parafilm M is utilized to cover vessels containing sample material, the extraction procedure needs to be conducted on a 1-cm2 piece, andtriplicates of the extractate shall be subjected to LAL analysis.14.2 Program microtiter plate well locations into plate reader software in acco
