ASTM E2657-09
(Test Method)Standard Test Method for Determination of Endotoxin Concentrations in Water-Miscible Metalworking Fluids
Standard Test Method for Determination of Endotoxin Concentrations in Water-Miscible Metalworking Fluids
SIGNIFICANCE AND USE
The determination of endotoxin concentrations in metalworking fluids is a parameter that can be used in decision-making for prudent fluid management practices (fluid draining, cleaning, recharging or biocide dosages).
This test method provides a test method for analysts who perform quantitative endotoxin analyses of water-miscible metalworking fluids.
SCOPE
1.1 This test method covers quantitative methods for the sampling and determination of bacterial endotoxin concentrations in water miscible metalworking fluids (MWF).
1.2 Users of this test method need to be familiar with the handling of MWF.
1.3 This test method gives an estimate of the endotoxin concentration in the sampled MWF.
1.4 This test method replaces E 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 responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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An American National Standard
Designation:E2657–09
Standard Test Method for
Determination of Endotoxin Concentrations in Water-
Miscible Metalworking Fluids
This standard is issued under the fixed designation E2657; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2.2 Government Standard:
29 CFR 1910.1450 Occupational Exposure to Hazardous
1.1 This test method covers quantitative methods for the
Chemicals in Laboratories
sampling and determination of bacterial endotoxin concentra-
29 CFR 1910.1000 Air Contaminants
tions in water miscible metalworking fluids (MWF).
1.2 Users of this test method need to be familiar with the
handling of MWF.
2.3 Other Documents:
1.3 This test method gives an estimate of the endotoxin
Criteria Document for a Recommended Standard: Occupa-
concentration in the sampled MWF.
tional Exposure to Metalworking Fluids, 1998
1.4 This test method replaces E2250.
NIOSH Manual of Analytical Methods (NMAM), 4th ed.,
1.5 This test method seeks to minimize inter-laboratory
Eller and Cassinelli, Eds., 1994
variation of endotoxin data but does not ensure uniformity of
results.
3. Terminology
1.6 This standard does not purport to address all of the
3.1 Definitions of Terms Specific to This Standard:
safety concerns, if any, associated with its use. It is the
3.1.1 control standard endotoxin (CSE), n—a purified
responsibility of the user of this standard to establish appro-
preparation of endotoxin based on the USPReference Standard
priate safety and health practices and determine the applica-
Endotoxin (RSE); used in laboratories to prepare standard
bility of regulatory limitations prior to use.
solutions.
2. Referenced Documents 3.1.2 endotoxin, n—a lipopolysaccharide derived from the
outer membrane of Gram-negative bacteria.
2.1 ASTM Standards:
3.1.3 endotoxin unit (EU), n—a biological potency unit
D2881 Classification for Metal Working Fluids and Related
equivalent to the FDA Reference Standard Endotoxin (RSE).
Materials
3.1.3.1 Discussion—The current RSE (EC-6) is equivalent
D4840 Guide for Sample Chain-of-Custody Procedures
to 1ng = 10 EU.
E1497 Practice for Selection and Safe Use of Water-
3.1.4 geometric mean (GM), n—the central tendency of a
Miscible and Straight Oil Metal Removal Fluids
set of numbers expressed as the nth root of their product.
E1542 Terminology Relating to Occupational Health and
3.1.5 geometric standard deviation (GSD), n—the spread of
Safety
data in a set of numbers expressed as a geometric mean.
3.1.6 gram-negative bacteria, n—prokaryotic cells that
This test method is under the jurisdiction of ASTM Committee E34 on
have a complex cell wall structure that stains characteristically
Occupational Health and Safety and is the direct responsibility of Subcommittee
when subjected to the differential Gram staining procedure.
E34.50 on Health and Safety Standards for Metal Working Fluids.
Current edition approved May 1, 2009. Published June 2009. DOI: 10.1520/
E2657-09.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
Standards volume information, refer to the standard’s Document Summary page on 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
the ASTM website. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2657–09
3.1.7 inhibition/enhancement phenomenon, n—conditions 5.2 This test method provides a test method for analysts
or artifacts in sample solutions that cause endotoxin concen- whoperformquantitativeendotoxinanalysesofwater-miscible
tration data from LAL assays to be less than or more than the metalworking fluids.
concentration of endotoxin actually present in a given aqueous
sample.
6. Interferences
3.1.8 Limulus amebocyte lysate (LAL) assay, n—a biologi-
6.1 Data from samples analyzed by LALmethodologies are
cal assay dependent on a series of cascading enzyme reactions
prone to variations due to batch differences in lysate
that occur when Limulus blood cell (amebocyte) lysate com-
composition/processing, non-optimal pH and temperatures of
bines with endotoxin.
assay solutions.
3.1.9 metalworking fluid (MWF), n—any fluid used for the
6.2 In the event that the phenomenon of inhibition/
purpose of cooling or treating metal surfaces during metal
enhancement influences this test method, endotoxin concentra-
removal, metal forming or surface protection or preservation.
tion data will be less than or more than actual concentrations
3.1.10 metal removal fluid (MRF), n—any fluid in the
present in a given metalworking fluid sample.
subclass of metalworking fluids used to cut, or otherwise take
6.3 LALassaysarehighlyinfluencedbytheskill/experience
away material or piece of stock.
level of the analyst.
3.1.10.1 Discussion—Metal removal fluids include straight
or neat oils (see D2881), not intended for further dilution with
7. Apparatus
water, and water miscible soluble oils, semisynthetics and
synthetics, which are intended to be diluted with water before 7.1 Sampling:
use. Metal removal fluids become contaminated during use in
7.1.1 Sample Collection Container, pyrogen-free, wide-
the workplace with a variety of workplace substances includ-
mouth, stainless steel sealable container, at least 100 mL
ing, but not limited to, abrasive particles, tramp oils, cleaners,
capacity.
dirt, metal fines and shavings, dissolved metal and hard water
7.1.2 Glass Pipet, pyrogen-free, 50 mL.
salts, bacteria, fungi, microbiological decay products, and
7.1.3 Battery-Powered Aspirator Unit (or Suction Bulb),
waste. These contaminants can cause changes in the lubricity
compatible with 100 mL glass pipet.
and cooling ability of the metal removal fluid as well as have
7.2 Extraction:
the potential to adversely affect the health and welfare of
7.2.1 Centrifuge, minimum rotational speed of 5000 rpm.
employees in contact with the contaminated metal removal
7.2.2 Ultrasonic Water Bath, ultrasonic/water bath appara-
fluid.
tus with a minimum peak frequency of 40 kHz with cavitation
3.1.11 operator-dependent assay, n—anassayperformedby
adjustment and thermostat control; use pyrogen-free glass
a technician in such a manner to cause significant influence(s)
containers only.
on the resultant data.
7.3 Reagents and Materials:
3.1.12 pyrogen-free (PF), adj—material(s) devoid of mea-
7.3.1 Control Standard Endotoxin (CSE), referenced to
surable endotoxin activity.
most current Federal Drug Administration (FDA) Reference
3.1.13 pyrogen-free water (PFW), n—processed water that
Standard Endotoxin (RSE).
is devoid of measurable endotoxin activity.
3.1.14 sensitivity range, n—a span of endotoxin measure- 7.3.2 Limulus Amebocyte Lysate (LAL), unexpired with
ments expressed as EU/mL or l.
stated potency.
7.3.3 Dilution water, pyrogen-free.
4. Summary of Test Method
7.4 Analysis:
4.1 Serial dilutions of CSE in PFW in borosilicate glass test 7.4.1 Incubating/Shaking Microplate Reader, spectrophoto-
tubes are prepared to construct a calibration curve.
metric at 405 nm.
4.2 The metalworking fluid sample is sonicated, centri-
7.4.2 Statistical Analysis Software Package for Microplate
fuged, and the supernatant retained.
Reader.
4.3 Triplicates of the sample supernatant, standard serial
7.4.3 Vortexer, variable speed.
dilutions,blanks,andpositivecontrolsolutionsaresubjectedto
7.4.4 Microtiter Plates, flat-bottomed, pyrogen-free, 96-
the kinetic chromogenic LAL assay.
well.
4.4 The reaction of Limulus amebocyte lysate with sample
7.4.5 Dilution Tubes, pyrogen-free, 13 by 100 mm.
endotoxin imparts a proportional yellow color to the analyte
7.4.6 Borosilicate Glass Test Tubes, pyrogen-free, screw
solution that is measured photometrically at 405 nm.
caps, 10 by 75 mm.
4.5 The measured endotoxin concentration is reported as
7.4.7 Single-Channel Micropipettor(s), 0.5-10 µL.
EU/mL.
7.4.8 Eight-Channel Micropipettor, 100 µL.
7.4.9 Pipet Tips, pyrogen-free, 300 µL.
5. Significance and Use
7.4.10 Glass Rod, pyrogen-free.
5.1 The determination of endotoxin concentrations in met-
7.4.11 Reagent Reservoir, pyrogen-free, 8-channel multipi-
alworking fluids is a parameter that can be used in decision-
pettor compatible.
making for prudent fluid management practices (fluid draining,
cleaning, recharging or biocide dosages). 7.4.12 Parafilm M
E2657–09
8. Hazards 11.1.2 Commercially-packaged labware that is nominally
described or labeled as sterile, sterilized, disinfected, or other-
8.1 Aerosols of endotoxin preparations pose a potential
wise identified as suitable for routine microbiological usage
respiratory hazard to susceptible laboratory personnel who are
only shall not be used in this test method, due to the possibility
directly involved with an endotoxin assay.
of the presence of residual endotoxin on critical labware
8.2 Inhalation or dermal exposure to metalworking fluids
surfaces.
pose potential health problems for personnel involved in MWF
11.1.3 Prior to use in this test method, non-pyrogen-free
sampling. Provision of personal protective equipment (PPE) in
glass or metal labware that will be used in LAL analyses shall
the form of respirators or protective clothing, or both, is
be subjected to the depyrogenation procedure described in
potentially indicated (see Practice E1497 and Criteria Docu-
Section 12.The analyst shall not use plastic labware, due to the
ment for a Recommended Standard: Occupational Exposure to
possibility of introducing non-specific assay interferences
Metalworking Fluids).
and/or causing container-related adsorption of endotoxin onto
8.3 Follow good laboratory procedures for worker protec-
surfaces.
tion and waste disposal, including 29 CFR 1910.1450.
8.4 Review material s
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