1、Designation: E2144 11 (Reapproved 2016) An 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 ind
2、icates the year oforiginal 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
3、 for the per-sonal 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 are
4、aexposure data of endotoxin 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 e
5、nsure uniformity of 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 method
6、s.1.6 In 1997, the 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 Assistan
7、t Secretary of Labor 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 assessment
8、s in facilities using 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 U
9、nitsThe values stated in SI units are to be regardedas standard. No other units of measurement are included in thisstandard.1.9 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 appr
10、o-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1356 Terminology Relating to Sampling and Analysis ofAtmospheresD4840 Guide for Sample Chain-of-Custody ProceduresD5337 Practice for Flow Rate Adju
11、stment of Personal Sam-pling PumpsD6629 Guide for Selection of Methods for Estimating SoilLoss by ErosionE1370 Guide for Air Sampling Strategies for Worker andWorkplace ProtectionE1497 Practice for Selection and Safe Use of Water-Miscible and Straight Oil Metal Removal FluidsE1542 Terminology Relati
12、ng to Occupational Health andSafety2.2 OSHA Standards:329 CFR 1910.1000 Air Contaminants1This 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 Standards for Metal Working Fluids.Cur
13、rent edition approved Oct. 1, 2016. Published October 2016. Originallyapproved in 2001. Last previous edition approved in 2011 as E2144 - 11. DOI:10.1520/E2144-11R16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual B
14、ook 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.govCopyright ASTM International, 100
15、 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States129 CFR 1910.1450 Occupational Exposure to HazardousChemicals in Laboratories2.3 Other Documents:Criteria Document for a Recommended Standard: Occupa-tional Exposure to Metalworking Fluids4NIOSH Manual of Analytical Meth
16、ods (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 and safety, refer to Terminology E1542.3.2 Definitions of Terms Specific to This
17、 Standard:3.2.1 endotoxin, npyrogenic high molar mass lipopolysac-charide (LPS) complex associated with the cell wall ofgram-negative bacteria.3.2.1.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endotoxins. Endotoxins are specifically de-tected through a Limulus Amoebocyte Lysate (
18、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 for sam-pling that is taken to the sampling site and handled in the samemanner as
19、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 the differential Gram staining procedure.3.2.5 Limulus amebocyte lysate (LAL) assa
20、y, 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 refers to straightoils and water-based fluids, such as soluble, semi-synthetic,a
21、nd 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 ascertain the concentration ofspecific constituents in the air in the persons br
22、eathing 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 volume of workplace air in a facility utilizingmetalworking fluids is drawn throug
23、h 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 subjected to quantitative endo-toxin analysis techniques. The measured endotoxin concent
24、ra-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 inhale them.Therefore, a consensus standard is needed to provide reliabledata on wor
25、kplace 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 of endotoxin exposure-response relation-ships.5.3 This practice facilitates compar
26、isons 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 variety of bio-molecular species and physicochemicalphenomena, such as pH, temperature,
27、 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. Apparatus7.1 Sampling:7.1.1 Sampling Unit, an apparatus consisting of a personalsampling
28、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 sampling pump withan on-board battery power source and a flow rate of 2.0 L/min(65
29、 %).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 support pad.7.1.1.4 Connective Tubing, flexible, appropriate inside di-ameter.7.1.1.5
30、 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 frequency of 40-kHz with cavitation adjust-ment and thermostat control.7.2.2 Vortex
31、 Mixer, general purpose with a minimum speedof 500 rpm.4Available 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.E2144 11 (2016)28.
32、Reagents and Materials8.1 Control Standard Endotoxin (CSE)Endotoxin prepa-rations used for calibration standards shall be referenced to theFederal Drug Administration (FDA) Reference Standard En-dotoxin (RSE), which is presently EC-6 RSE. Calibrationstandards data and corresponding regression data a
33、re expressedin EU.8.2 Endotoxin detection reagents, utilized in accordancewith manufacturers directions.9. Hazards9.1 Aerosols of endotoxin preparations pose a potentialrespiratory hazard to susceptible laboratory personnel who aredirectly involved with an endotoxin assay.9.2 Follow good laboratory
34、procedures for worker protec-tion and waste disposal, including 29 CFR 1910.1450.9.3 Inhalation or dermal exposure to metalworking fluidsmay pose health problems for personnel involved in aerosolsampling. Provision of personal protective equipment (PPE) inthe form of respirators or protective clothi
35、ng, or both, may beindicated (see Practice E1497 and Criteria Document for aRecommended Standard: Occupational Exposure to Metal-working Fluids).9.4 Review material safety data sheets (MSDS) for materi-als in use at a facility to identify potential hazards to determineappropriate personal protective
36、 equipment (see 29 CFR1910.1000).10. Pump Calibration and Standardization10.1 Calibrate the airflow rate of the sampling pump on sitebefore each sampling period. The final flow rate shall bedetermined after sample collection is complete. Samplesshould be voided if flow rate changes significantly dur
37、ing thesample period.10.2 Maintenance and repairs of the sampling and analyticalequipment should be performed according to the recommen-dations of the manufacturer and should be documented inmaintenance records.10.3 Airflow rate procedures shall be performed in accor-dance with Practice D5337 or NMA
38、M 1994 Calibration ofPersonal Sampling Pumps.11. Sampling11.1 Plan air-sampling strategies for metalworking fluidaerosol endotoxin analysis using Guide 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 cas
39、sette, 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 sample shall link to the following information:date of sample, location of work operation, sample volume,investigator/worker code, and any other pe
40、rtinent 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 Sampler: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 perso
41、nal sampling pumpto 2.0 L/min (6 5 %) and record room temperature.11.3.3 Attach the filter cassette in the breathing zone of theindividual being tested, activate the personal sampling pumpand record the starting time. Total sampling duration shall bedetermined on the basis of partial or total workda
42、y shifts ordiscrete work activities.11.3.4 Deactivate the sampling pump after the samplingperiod and record the stopping time, temperature, and anyunusual 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 eache
43、nd.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 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, shi
44、p 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 Guide 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).
45、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 procedures on the same day that the filters areremoved from their cassettes. Samples shall be allowed towarm to room temperature and the entire extr
46、action procedureshall be conducted at room temperature (25 6 2C).13.2 Aseptically remove the filter and support pad from thecassette with depyrogenated tweezers 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 tes
47、t 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 extract 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 extra
48、ct,and if necessary, adjust the sample extract to pH 7.5 withpyrogen-free sodium hydroxide or hydrochloric acid.14. Procedure14.1 Analysis of sample extracts with endotoxin detectionreagents shall be performed in accordance with manufacturersdirections.E2144 11 (2016)315. Quality Assurance15.1 Ensur
49、e validation and maintenance procedures havebeen conducted in accordance with spectrophotometer manu-facturers directions.15.2 The correctness of software calculations shall 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
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