ASTM E2275-03(2008)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E2275 − 03(Reapproved 2008)
Standard Practice for
Evaluating Water-Miscible Metalworking Fluid Bioresistance
and Antimicrobial Pesticide Performance
This standard is issued under the fixed designation E2275; 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 D1067 Test Methods for Acidity or Alkalinity of Water
D1193 Specification for Reagent Water
1.1 This practice addresses the evaluation of the relative
D3342 Test Method for Dispersion Stability of New (Un-
inherent bioresistance of water-miscible metalworking fluids,
used) Rolling Oil Dispersions in Water
the bioresistance attributable to augmentation with antimicro-
D3519 Test Method for Foam in Aqueous Media (Blender
bial pesticides or both. It replaces Methods D3946 and E686.
Test)
1.2 In this practice relative bioresistance is determined by
D3601 Test Method for Foam In Aqueous Media (Bottle
challenging metalworking fluids with a biological inoculum
Test)
that may either be characterized (comprised of one or more
D4012 Test Method forAdenosineTriphosphate (ATP) Con-
known biological cultures) or uncharacterized (comprised of
tent of Microorganisms in Water
biologically contaminated metalworking fluid or one or more
D4627 Test Method for Iron Chip Corrosion for Water–Di-
unidentified isolates from deteriorated metalworking fluid).
lutable Metalworking Fluids
Challenged fluid bioresistance is defined in terms of resistance
D5465 Practice for Determining Microbial Colony Counts
to biomass increase, viable cell recovery increase, chemical
from Waters Analyzed by Plating Methods
property change, physical property change or some combina-
E70 Test Method for pH of Aqueous Solutions With the
tion thereof.
Glass Electrode
1.3 This practice is applicable to antimicrobial agents that E1326 GuideforEvaluatingNonconventionalMicrobiologi-
cal Tests Used for Enumerating Bacteria
are incorporated into either the metalworking fluid concentrate
or end-use dilution. It is also applicable to metalworking fluids E2169 Practice for Selecting Antimicrobial Pesticides for
Use in Water-Miscible Metalworking Fluids
that are formulated using non-microbicidal, inherently biore-
sistant components.
2.2 Other Standards:
4.027 Synthetic Hard Water
1.4 The values stated in SI units are to be regarded as the
9215A.6a Heterotrophic Plate Count Media, Plate Count
standard. The values given in parentheses are for information
Agar
only.
9216 Direct Total Microbial Count
1.5 This standard does not purport to address all of the
Microbiological Test <71>
safety concerns, if any, associated with its use. It is the
2.3 Government Standard:
responsibility of the user of this standard to establish appro-
40 CFR 156 Labeling Requirements for Pesticides and De-
priate safety and health practices and determine the applica-
vices
bility of regulatory limitations prior to use.
3. Terminology
2. Referenced Documents
3.1 Definitions:
2.1 ASTM Standards:
3.1.1 active ingredient, n—the chemical component or com-
D888 Test Methods for Dissolved Oxygen in Water
ponents of an antimicrobial pesticide that provides its micro-
bicidal performance.
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2008. Published May 2008. Originally AOAC International Methods of Analysis, AOAC International, Gaithersburg,
ϵ1
approved in 2003. Last previous edition approved in 2003 as E2275 – 03 . DOI: MD.
10.1520/E2275-03R08. Available from American Public Health Association (APHA) Standard Meth-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or ods for the Examination of Water and Wastewater800IStreet,NWWashington,DC
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM 20001.
Standards volume information, refer to the standard’s Document Summary page on Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,
the ASTM website. MD 20852-1790, http://www.usp.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2275 − 03 (2008)
3.1.2 antimicrobial pesticide, n—chemical additive regis- 4.2.1 Shorter test periods are used to evaluate microbicide
teredunder40 CFR 152,forusetoinhibitgrowth,proliferation speed of kill and metalworking formulation initial bioresis-
or both of microorganisms. tance.
4.2.2 Longer test periods are used to evaluate metalworking
3.1.3 as supplied, adj—antimicrobial pesticide finished
fluid formulation resistance to repeated challenges. For tests
product including the active ingredient(s), solvent and any
lasting longer than one-week, 10 to 80 % of the fluid is
additional inactive ingredients.
exchanged weekly with fresh fluid before the additional
3.1.4 biocide, n—any chemical intended for use to kill
challenge. The percentage of fluid exchange should reflect
organisms.
anticipated fluid turnover rates in fluid’s end-use application.
3.1.5 bioresistant, adj—ability to withstand biological at-
4.3 Bioresistance is determined as the test fluid’s relative
tack.
abilitytopreventtheproliferationofchallengemicrobes,retain
3.1.5.1 Discussion—Bioresistant, or recalcitrant, chemicals
its original chemical or physical properties of some combina-
are not readily metabolized by microorganisms.
tion of the above. The bioresistance of test formulations is
3.1.6 biostatic, adj—able to prevent existing microbial con-
defined relative to that of a benchmark or control formulation.
taminants from growing or proliferating, but unable to kill
them.
5. Significance and Use
3.1.6.1 Discussion—Biostatic additives may be registered
5.1 This practice provides laboratory procedures for rating
antimicrobial pesticides or unregistered chemicals with other
the relative bioresistance of metalworking fluid formulations,
performance properties. The difference between biocidal and
for determining the need for microbicide addition prior to or
biostatic performance may be attributed to dose, chemistry or
during fluid use in metalworking systems and for evaluating
both.
microbicide performance. General considerations for microbi-
3.1.7 dose, n—concentration of antimicrobial pesticide
cide selection are provided in Practice E2169.
added to treated solution.
3.1.7.1 Discussion—Dose is generally expressed as either 5.2 The factors affecting challenge population numbers,
ppm active ingredient (a.i.) or ppm as supplied (a.s.). taxonomic diversity, physiological state, inoculation frequency
and biodeterioration effects in recirculating metalworking fluid
3.1.8 inactive ingredient, n—component of antimicrobial
systems are varied and only partially understood. Conse-
pesticide that is not directly responsible for the pesticide’s
quently, the results of tests completed in accordance with this
antimicrobial performance.
practice should be used only to compare the relative perfor-
3.1.8.1 Discussion—Inactive ingredients may include, but
mance of products or microbicide treatments included in a test
are not limited to solvents and chemicals that improve the
series. Results should not be construed as predicting actual
pesticide’s non-biocidal performance properties, such as mis-
field performance.
cibility and reactivity with non-target molecules in the treated
material.
6. Apparatus
3.1.9 minimum inhibitory concentration (MIC), n—lowest
6.1 Air Supply, air provided at no more than 110 kPa.
treatment-dose that will prevent test population from growing,
proliferating or otherwise contributing to biodeterioration.
NOTE 1—Any air source that is free of organic vapors, organic matter
or other objectionable material may be used. Sterile air need not be used
3.2 Abbreviations:
for the uncharacterized inoculum, but shall be used for the characterized
3.2.1 a.i.—active ingredient
inoculum. If necessary, air may be sterilized either by inserting, in series,
3.2.2 a.s.—as supplied
twocommerciallyavailablein-linesterilefiltersdesignedforthispurpose.
3.2.3 ATCC—american type culture collection
Alternatively an in-line filter may be prepared as follows: Pack two 150
3.2.4 CFU—colony forming unit mm long drying tubes (bulb-type) loosely with borosilicate glass wool in
series with neoprene stoppers, glass tubing and neoprene tubing. Wrap
loosely in aluminum foil and steam sterilize at 103 to 138 kPa (15 to 20
4. Summary of Practice
psi) for 30 min or dry heat sterilize at 160°C for 2 h. Cool to room
4.1 End-use dilutions of one or more water-miscible metal- temperature while wrapped. Insert into air line with bulbs on upstream
side. Whether using a commercial or fabricated filter, average lifetime in
working fluids are dispensed into microcosms. The fluids may
continuous use is two weeks. Discard sooner if upstream filter becomes
be fresh or aged, dosed with one or more antimicrobial
wet or contaminated with oil.
pesticides or undosed. Microcosms are challenged with either
6.2 Aquarium Tubing, 6.35 mm (0.25 in.) diameter, silicone
uncharacterized or characterized biological inocula. After in-
or vinyl.
oculation, microcosms are aerated either continuously or peri-
odically to simulate recirculation conditions in coolant sys-
6.3 Autoclave, with both steam cycle (80 to 100°C) and
tems. Chips may also be added to microcosms to simulate chip
sterilization cycle (15 min at ≥ 121°C) capability.
accumulation in coolant systems.
6.4 Adjustable Volume Pipetters, with sterile disposable
4.2 After inoculation, fluid samples are drawn from each
tips. Pipetters will be used to deliver 1.0 µL to 2 mL volumes.
microcosm periodically and tested for the parameters of
6.5 Glassware:
interest, including but not limited to microbial viable counts.
Depending on the test objectives, the test duration may range
NOTE 2—Sterile laboratory ware or sterile disposable laboratory ware
from 24 h to three months. should be used according to standard microbiological practice.
E2275 − 03 (2008)
concentration range. If the test objective is to evaluate microbicide
6.5.1 Glass Tubing, 6.35 mm (0.25 in.) i.d., cut into 15 cm
v
performance in multiple metalworking fluid formulations, a 5 % ( ⁄v)
lengths with ends fire-polished.
end-use dilution is appropriate.
6.5.2 French Square Bottles, 960 mL, with metal cap.
7.2 Materials:
NOTE 3—Alternatively, 1 L capacity canning jars may be used.
7.2.1 Inoculum—The microbial inoculum may vary accord-
6.5.3 Pipetes, Bacteriological, 10 and 2.2 mL.
ingtotheuser’srequirements.Itmaybeeithercharacterizedor
6.6 Incubator,capableofmaintainingatemperatureof25 6 uncharacterized. The challenge population should be accli-
2°C. mated to the metalworking fluid before being used in this
method. Acclimatization shall be achieved by growing the
NOTE 4—Although an incubator is preferred, incubation may be
challenge in the end-use dilution, negative-control metalwork-
performed at ambient room temperature.
ing fluid formulation.
6.7 Manifold, aquarium style, multi-valve.
7.2.1.1 Prepare an uncharacterized inoculum by adding 50
NOTE5—Thenumberofmanifoldsandvalvespermanifoldwilldepend
mL of spoiled metalworking fluid to 850 mL of freshly
on the number of microcosms in the test array. Air for each microcosm
prepared end-use dilution, negative-control metalworking
shall be supplied through a single air valve. Where used, air sterilization
fluid. Aerate at 25 6 2°C or at ambient room temperature for
filters shall be placed between the air valve and microcosm aeration tube.
24 h or until the microbial viable count reaches 10 CFU ·
6.8 Metal Punch, 1 cm diameter.
-1
mL . Replace 800 mL of this fluid with freshly prepared
portion of the negative-control fluid. Repeat the aeration and
7. Reagents and Materials
metalworking fluid replacement procedure for a minimum of
7.1 Reagents:
three cycles before using the preparation as an inoculum.
7.1.1 Purity of Reagents—Reagent grade chemicals shall be
7.2.1.2 Prepare a characterized inoculum by using standard
used in all tests. Unless otherwise indicated, it is intended that
microbiological techniques to isolate, maintain and identify
all reagents conform to the specifications of the Committee on
specific microbes from spoiled metalworking fluid. Alterna-
Analytical Reagents of the American Chemical Society where
tively, cultures of specific interest may be obtained from a
such specifications are available.
commercial type culture collection. Examples of commercial
7.1.2 Water Purity—Unless otherwise indicated, references
cultures that may be used are: Aeromonas hydrophila
to water shall be understood to mean reagent water as defined
(ATCC 13444), Candida albicans (ATCC 752), Desulfovibrio
by Type III of Specification D1193.
desulfuricans (ATCC 7757), Escherichia coli (ATCC 8739),
7.1.3 Antimicrobial Pesticide(s):
Flavobacterium ferrugineum (ATCC 13524), Fusarium ox-
ysporum (ATCC 7601), Klebsiella pneumonia (ATCC 13883),
NOTE 6—The measurement of antimicrobial pesticide (microbicide)
efficacy in a medium as complex as metalworking fluid is relative, not
Mycobacterium immunogenum (Rossmoore strain), Proteus
absolute. Consequently, when this method is used to evaluate microbicide
mirabilis (ATCC 4675), Pseudomonas aeruginosa
performance (8.3 or 8.4), it is prudent to always evaluate at least two
(ATCC 8689), Pseudomonas oleovorans (ATCC 8062) and
antimicrobial treatments. Preferably one treatment should serve as a
Saccharomyces cerevisiae (ATCC 2338). Before using a char-
positive control; its efficacy in the test system having been established
acterized inoculum for metalworking fluid bioresistance test-
previously.
ing, acclimate the inoculum following the procedure described
7.1.4 Metalworking Fluid(s):
for an uncharacterized inoculum (7.2.1.1). Warning—
NOTE 7—The number of metalworking fluids available is almost
Microbes recovered from metalworking fluids may be patho-
limitless. Recommendations for the use of any particular fluid cannot be
genic. Do not pipet by mouth.
made. If the primary intent is to evaluate the general efficacy of the
microbicide(s) being tested, then it/they should be tested in various types
NOTE 9—As more bioresistant metalworking fluid formulations are
of formulations. If the primary intent is to protect a particular formulation,
developed, microbicide-free control fluid may not support microbial
then a microbicide-free version of that formulation should be used as the
growth at normal end-use
...