1、Designation: E2144 11An American National StandardStandard Practice forPersonal Sampling and Analysis of Endotoxin inMetalworking Fluid Aerosols in Workplace Atmospheres1This standard is issued under the fixed designation E2144; the number immediately following the designation indicates the year ofo
2、riginal 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 practice covers quantitative methods for the per-sonal
3、sampling and determination of bacterial endotoxin con-centrations in poly-disperse metalworking fluid aerosols inworkplace atmospheres. Users should have fundamentalknowledge of microbiological techniques and endotoxin test-ing.1.2 Users of this practice may obtain personal or areaexposure data of e
4、ndotoxin in metalworking fluid aerosols,either on a short-term or full-shift basis in workplace atmo-spheres.1.3 This practice gives an estimate of the endotoxin con-centration of the sampled atmosphere.1.4 This practice seeks to minimize inter laboratory varia-tion but does not ensure uniformity of
5、 results.1.5 It is anticipated that this practice will facilitate interlaboratory comparisons of airborne endotoxin data from met-alworking fluid atmospheres, particularly metal removal fluidatmospheres, by providing a basis for endotoxin sampling,extraction, and analytical methods.1.6 In 1997, the
6、Occupational Safety and Health Adminis-tration (OSHA) empanelled a Standards Advisory Committeeto make recommendations to the Administration regardingmeasures that the Administration could take to improve thehealth of workers exposed to metalworking fluids. A report tothe Assistant Secretary of Labo
7、r for OSHA was submitted inJuly, 1999. Subcommittee E34.50 believes that the user com-munity would benefit significantly if a standard method wasdeveloped to give the community guidance on a methodologyfor the sampling and analysis of personal airborne endotoxinexposure assessments in facilities usi
8、ng water-miscible metalremoval fluids, based on the LAL assay or other endotoxindetection technologies as they become available.1.7 This practice does not attempt to set or imply limits forpersonal exposure to endotoxin in metalworking fluid aerosolsin workplace environments.1.8 This standard does n
9、ot 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 determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standar
10、ds:2D1356 Terminology Relating to Sampling and Analysis ofAtmospheresD4840 Guide for Sample Chain-of-Custody ProceduresD5337 Practice for Flow Rate Adjustment of PersonalSampling PumpsD6629 Guide for Selection of Methods for Estimating SoilLoss by ErosionE1370 Guide for Air Sampling Strategies for W
11、orker andWorkplace ProtectionE1497 Practice for Selection and Safe Use of Water-Miscible and Straight Oil Metal Removal FluidsE1542 Terminology Relating to Occupational Health andSafety2.2 OSHA Standards:329 CFR 1910.1000 Air Contaminants29 CFR 1910.1450 Occupational Exposure to HazardousChemicals i
12、n Laboratories2.3 Other Documents:Criteria Document for a Recommended Standard: Occupa-tional Exposure to Metalworking Fluids41This practice is under the jurisdiction of ASTM Committee E34 on Occupa-tional Health and Safety and is the direct responsibility of Subcommittee E34.50 onHealth and Safety
13、Standards for Metal Working Fluids.Current edition approved Dec. 1, 2011. Published December 2011. Originallyapproved in 2001. Last previous edition approved in 2007 as E2144 - 01(2007).DOI: 10.1520/E2144-11.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Custome
14、r 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 Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.acces
15、s.gpo.gov4Available from U.S. Department of Health and Human Services, Public HealthService, Centers for Disease Control and Prevention, National Institute for Occu-pational Safety and Health, 4676 Columbia Pkwy., Cincinnati, OH 45226.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700
16、, West Conshohocken, PA 19428-2959, United States.NIOSH Manual of Analytical Methods (NMAM)43. Terminology3.1 For definitions of terms in this practice relating tosampling and analysis of atmospheres, refer to TerminologyD1356. For definitions of terms in this practice relating tooccupational health
17、 and safety, refer to Terminology E1542.3.2 Definitions of Terms Specific to This Standard:3.2.1 endotoxin, npyrogenic high molar mass lipopoly-saccharide (LPS) complex associated with the cell wall ofgram-negative bacteria.3.2.1.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endoto
18、xins. Endotoxins are specifically de-tected through a Limulus Amoebocyte Lysate (LAL) test.3.2.2 endotoxin unit (EU), na biological potency unitequivalent to the FDA Reference Standard Endotoxin (RSE).Currently, EC-6 is equivalent to 0.1 ng 3D 1 EU.3.2.3 field blank, nfilter/cassette unit prepared f
19、or sam-pling that is taken to the sampling site and handled in the samemanner as the analytical filter/cassette unit, but that is not a partof the sampling process.3.2.4 Gram-negative bacteria, nprokaryotic cells thathave a complex cell-wall structure that stain characteristicallywhen subjected to t
20、he differential Gram staining procedure.3.2.5 Limulus amebocyte lysate (LAL) assay, na biologi-cal assay that detects endotoxin.3.2.6 metal removal fluids, nthe subset of metal workingfluids that are used for wet machining or grinding to producethe finished part.3.2.6.1 DiscussionThe term most often
21、 refers to straightoils and water-based fluids, such as soluble, semi-synthetic,and synthetic fluids.3.2.7 onset time, ntime required for a change of 200 mOD(optical density) units relative to the initial OD value.3.2.8 personal sampler, na portable sampling instrumentthat is attached to a person to
22、 ascertain the concentration ofspecific constituents in the air in the persons breathing zone.3.2.9 pyrogen-free, adjmaterial(s) devoid of measurableendotoxin activity.3.2.10 pyrogen-free water (PFW), nprocessed water thatis devoid of measurable endotoxin activity.4. Summary of Practice4.1 A known v
23、olume of workplace air in a facility utilizingmetalworking fluids is drawn through a sample filter cassetteunit.4.2 The sample filter is extracted into a pyrogen-free solu-tion to quantitatively release endotoxin absorbed from col-lected metalworking fluid aerosol.4.3 The extract solution is subject
24、ed to quantitative endo-toxin analysis techniques. The measured endotoxin concentra-tion is reported in terms of endotoxin potency units per unitvolume of air sampled.5. Significance and Use5.1 Endotoxins in metalworking fluid aerosols present po-tential respiratory health hazards to workers who inh
25、ale them.Therefore, a consensus standard is needed to provide reliabledata on workplace airborne endotoxin concentrations wheremetalworking fluids are used.5.2 This practice for measuring airborne endotoxin concen-trations in metalworking fluid atmospheres will help to foster abetter understanding o
26、f endotoxin exposure-response relation-ships.5.3 This practice facilitates comparisons of inter laboratorydata from methods and field investigative studies.6. Interferences6.1 Airborne endotoxin measurements resulting from use ofLAL reagents are subject to inhibition/enhancement effectsfrom a variet
27、y of bio-molecular species and physicochemicalphenomena, such as pH, temperature, filter matrix effects,cationic concentrations, LAL-reactive materials (LRM), en-zyme influences, and lysate composition variability and sensi-tivity (a function of different lysate processing methodologies).7. Apparatu
28、s7.1 Sampling:7.1.1 Sampling Unit, an apparatus consisting of a personalsampling pump, a 37-mm glass fiber filter, a two-piece,closed-face plastic cassette, and flexible connecting tubingbetween the personal sampling pump and the attached cassette/filter unit.7.1.1.1 Pump, a constant-flow personal s
29、ampling pump withan on-board battery power source and a flow rate of 2.0 L/min(65 %).7.1.1.2 Filter Cassette, pyrogen-free, closed-faced, two-piece polystyrene filter holder with 4 mm inlet and outlet, withcaps.7.1.1.3 Filter (Membrane), pyrogen-free, glass fiber, 37-mmdiameter with a cellulose supp
30、ort pad.7.1.1.4 Connective Tubing, flexible, appropriate inside di-ameter.7.1.1.5 Soap-bubble Meter, a primary standard used forsampler flow rate calibration.NOTE 1An alternative primary standard is acceptable.7.2 Extraction:7.2.1 Sonicator Bath, ultrasonic/water bath apparatus with aminimum peak fr
31、equency of 40-kHz with cavitation adjust-ment and thermostat control.7.2.2 Vortex Mixer, general purpose with a minimum speedof 500 rpm.8. Reagents and Materials8.1 Control Standard Endotoxin (CSE)Endotoxin prepa-rations used for calibration standards shall be referenced to theFederal Drug Administr
32、ation (FDA) Reference Standard En-dotoxin (RSE), which is presently EC-6 RSE. Calibrationstandards data and corresponding regression data are expressedin EU.8.2 Endotoxin detection reagents, utilized in accordancewith manufacturers directions.9. Hazards9.1 Aerosols of endotoxin preparations pose a p
33、otentialrespiratory hazard to susceptible laboratory personnel who aredirectly involved with an endotoxin assay.E2144 1129.2 Follow good laboratory procedures for worker protec-tion and waste disposal, including 29 CFR 1910.1450.9.3 Inhalation or dermal exposure to metalworking fluidsmay pose health
34、 problems for personnel involved in aerosolsampling. Provision of personal protective equipment (PPE) inthe form of respirators or protective clothing, or both, may beindicated (see Practice E1497 and Criteria Document for aRecommended Standard: Occupational Exposure to Metal-working Fluids).9.4 Rev
35、iew material safety data sheets (MSDS) for materi-als in use at a facility to identify potential hazards to determineappropriate personal protective equipment (see 29 CFR1910.1000).10. Pump Calibration and Standardization10.1 Calibrate the airflow rate of the sampling pump on sitebefore each samplin
36、g period. The final flow rate shall bedetermined after sample collection is complete. Samplesshould be voided if flow rate changes significantly during thesample period.10.2 Maintenance and repairs of the sampling and analyticalequipment should be performed according to the recommen-dations of the m
37、anufacturer and should be documented inmaintenance records.10.3 Airflow rate procedures shall be performed in accor-dance with Practice D5337 or NMAM 1994 Calibration ofPersonal Sampling Pumps.11. Sampling11.1 Plan air-sampling strategies for metalworking fluidaerosol endotoxin analysis using Guide
38、E1370.11.2 Filter/Cassette Unit Set-up:11.2.1 Aseptically transfer a glass fiber filter and supportpad to a closed-face, three-piece polystyrene cassette, and thenassemble the cassette and seal the perimeter seams with PTFEtape.11.2.2 Affix a label to the cassette with a unique sampleidentifier. The
39、 sample shall link to the following information:date of sample, location of work operation, sample volume,investigator/worker code, and any other pertinent information.11.2.3 Store and transport at least one unused (blank)filter/cassette unit from the same lot as described in 11.2.1.11.3 Personal Sa
40、mpler:11.3.1 Uncap filter/cassette unit and attach to calibratedpersonal sampler pump with flexible tubing.11.3.2 Set the sampling rate of the personal sampling pumpto 2.0 L/min (65 %) and record room temperature.11.3.3 Attach the filter cassette in the breathing zone of theindividual being tested,
41、activate the personal sampling pumpand record the starting time. Total sampling duration shall bedetermined on the basis of partial or total workday shifts ordiscrete work activities.11.3.4 Deactivate the sampling pump after the samplingperiod and record the stopping time, temperature, and anyunusua
42、l conditions in the sampling area that could bias theoutcome of the sampling procedure.11.3.5 Remove the used filter/cassette unit and cap at eachend.12. Storage and Shipment12.1 Store the used labeled filter/cassette unit(s) in a suit-able container at 4 6 2C until shipped or analyzed. Do notfreeze
43、 sample at any time.12.2 Samples should be shipped via overnight delivery. Ifthe shipment will take more than 24 h to arrive at itsdestination, ship the samples in a suitable container at 4 6 2C.12.3 Maintain procedures for sample custody in accordancewith accepted chain of custody procedures (see G
44、uide D4840).12.4 Upon receiving the used filter/cassette unit(s), labora-tory personnel shall record the date and time of receipt of thesample(s). Prior to extraction, samples shall be stored at 4 62C. Do not freeze.13. Extraction13.1 The analyst shall subject sample and blank filters toextraction p
45、rocedures on the same day that the filters areremoved from their cassettes. Samples shall be allowed towarm to room temperature and the entire extraction procedureshall be conducted at room temperature (25 6 2C).13.2 Aseptically remove the filter and support pad from thecassette with depyrogenated t
46、weezers and place filter into apyrogen-free test tube. Discard support pad.13.3 Add an extraction volume of 20 mL of pyrogen-freewater into the test tube which is then capped and bath sonicatedat a minimum peak frequency of 40 kHz for1hat256 2C(or shake vigorously for 1 h).13.4 Centrifuge the extrac
47、t at 1000 g for at least 15 min.13.5 Transfer the supernatant into a pyrogen-free test tube.13.6 Determine the pH of an aliquot of the sample extract,and if necessary, adjust the sample extract to pH 7.5 withpyrogen-free sodium hydroxide or hydrochloric acid.14. Procedure14.1 Analysis of sample extr
48、acts with endotoxin detectionreagents shall be performed in accordance with manufacturersdirections.15. Quality Assurance15.1 Ensure validation and maintenance procedures havebeen conducted in accordance with spectrophotometer manu-facturers directions.15.2 The correctness of software calculations s
49、hall be vali-dated at least annually by checking selected generated datawith other software or calculators.15.3 Individuals who perform endotoxin assays shall beappropriately trained. Good laboratory quality assurance pro-cedures should be in place.15.4 Ensure that linearity of standard curve, spike recoveryand other quality control measures meet manufacturers speci-fications.16. Calculation or Interpretation of Results16.1 Endotoxin concentration of standards and samplesshall be determined in accordance with kit manufacturersdirections.16.2 Resultant endotox
