ASTM E645-97
(Test Method)Standard Test Method for Efficacy of Microbicides Used in Cooling Systems
Standard Test Method for Efficacy of Microbicides Used in Cooling Systems
SCOPE
1.1 This test method outlines a procedure for evaluating the efficacy of microbicides (algicides, bactericides, and fungicides) that will be used for controlling microbial growth in cooling water systems. The microbicides will be evaluated using simulated or real cooling tower water against microbes from cooling water, microbiological deposits (biofilms) from operating cooling systems, or microorganisms known to contaminate cooling water systems, or a combination thereof. This test method should be performed by individuals familiar with microbiological techniques.
1.2 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)
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Designation: E 645 – 97
Standard Test Method for
Efficacy of Microbicides Used in Cooling Systems
This standard is issued under the fixed designation E 645; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.2 bactericides, n—an agent that kills bacteria. This term
is applied to chemical agents that kill all bacteria, but not
1.1 This test method outlines a procedure for evaluating the
necessarily bacterial spores.
efficacy of microbicides (algicides, bactericides, and fungi-
3.1.3 biofilm, n—an accumulation of cells immobilized on a
cides) that will be used for controlling microbial growth in
substratum and frequently embedded in an organic polymer
cooling water systems. The microbicides will be evaluated
matrix of microbial origin.
using simulated or real cooling tower water against microbes
3.1.4 biofouling, n—the unwanted accumulation of cells
from cooling water, microbiological deposits (biofilms) from
and their products on surfaces. Many times this accumulation
operating cooling systems, or microorganisms known to con-
is accompanied by deposition of organic and inorganic mate-
taminate cooling water systems, or a combination thereof. This
rial.
test method should be performed by individuals familiar with
3.1.5 cooling system, n—an assemblage of equipment for
microbiological techniques.
the removal of heat from processes or equipment, or both. The
1.2 This standard does not purport to address all of the
most common medium used for removal or transfer of heat is
safety concerns, if any, associated with its use. It is the
water. The heated water then can be discharged into a receiving
responsibility of the user of this standard to establish appro-
body (once through cooling system) or it can be cooled and
priate safety and health practices and determine the applica-
reused (recirculating cooling system).
bility of regulatory limitations prior to use.
3.1.6 cooling tower, n—a structure used to dissipate heat in
2. Referenced Documents open recirculating cooling systems.
3.1.7 cooling water, n—medium used to transfer heat in
2.1 ASTM Standards:
cooling systems.
D 3731 Practices for Measurement of Chlorophyll Content
3.1.8 fungicides, n—an agent that kills fungi, both fungal
of Algae in Surface Waters
vegetative cells and spores. This term is applied mostly to
D 4412 Test Methods for Sulfate-Reducing Bacteria in
chemical agents.
Water and Water-Formed Deposits
3.1.9 microbicides, n—an agent that kills microbes: bacte-
E 1054 Practices for Evaluating Inactivators of Antimicro-
rial vegetative cells, fungal vegetative cells and spores, algae,
bial Agents Used in Disinfectants, Sanitizer, Antiseptic, or
and protozoa. This term is applied to chemical agents that kill
Preserved Products
microbes.
E 1326 Guide for Evaluating Nonconventional Microbio-
logical Tests Used for Enumerating Bacteria
4. Summary of Test Method
E 1427 Guides for Selecting Test Methods to Determine the
4.1 Microbicides are evaluated against microbes under con-
Effectiveness of Antimicrobial Agents and Other Chemi-
ditions simulating a cooling water system. Microbicides at
cals for the Prevention, Inactivation, and Removal of
concentrations that are expected to control the microbes are
Biofilm
added to cooling water. At selected time periods, the amount of
3. Terminology microbes in the water are determined and compared to values
at the start of the experiment. Bacteria (aerobic and anaerobic),
3.1 Definitions of Terms Specific to This Standard:
fungi or algae, or both, may be detected by a number of
3.1.1 algicide, n—a substance that kills algae; unicellular
methods, such as plate counting, Most Probable Number
chlorophyll-containing plants.
(MPN), Adenosine-58-Triphosphate (ATP). The investigator
will determine the minimal microbicide concentration for
This test method is under the jurisdiction of ASTM Committee E-35 on
efficacy based upon laboratory registration needs.
Pesticides and is the direct responsibility of Subcommittee E35.15 on Antibacterial
and Antiviral Agents.
5. Significance and Use
Current edition approved April 10, 1997. Published June 1997. Originally
5.1 This test method determines potentially effective micro-
published as E 645 – 78. Last previous edition E 645 – 91.
Annual Book of ASTM Standards, Vol 11.05.
bicides for use in cooling water systems using cooling water
Annual Book of ASTM Standards, Vol 11.02.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 645
and deposits/biofilm obtained from the field. The addition of water or other solvent used in preparing the microbicide stock
deposits/biofilms addresses the need to include the major solutions. Reagent grade organic solvents should be used if
source of microorganisms in cooling water systems. Even with water is not a suitable diluent for a particular microbicide. If a
this addition, however, laboratory results may not be predictive solvent other than water is used, however, an additional control
of microbicide effectiveness in the field. This, in part, is due to that has solvent without any of the microbicide added to the
conditions in the field that effect microbicide efficacy and are cooling water sample should be used to demonstrate that the
hard to mimic in the laboratory, that is, blow down rate, chosen solvent has no appreciable effect on the test results.
addition of makeup water, water hardness, hydrocarbon leaks, 7.2 Purity of Water—All reference to water as a diluent or
pH, sediment loading, dissolved solids, microbes in slime, and reagent shall mean distilled water or water of equal purity,
deposits (biofilms) on surfaces. Another factor is the difficulty unless otherwise noted.
in enumerating microbes in the water due to the lack of 7.3 Culture Media:
adequate recoverable medium. Guidelines that address forma- 7.3.1 A general bacterial agar medium, such as Glucose
tion of and testing for surface-attached microbes (biofilms) Extract Agar, Tryptic Soy Agar, R2AAgar, and so forth, is used
may be found in Guides E 1427, while a guideline for for conducting bacterial counts on test samples. Other media,
unconventional measurement of microbes is found in Guide such as selective or differential types or procedures, such as
E 1326. ATP measurement, may be used to monitor the bacteria.
7.3.2 A general fungal medium, such as inhibitory mold
6. Apparatus
agar, Sabouraud dextrose agar, and so forth, is used to for
6.1 Balance—An analytical balance sensitive to 0.1 mg
conducting fungal counts on the samples. This medium must
should be employed to weigh the candidate microbicide to be
be able to inhibit the growth of bacteria.
used in the preparation of stock solutions. 7.3.3 Bristol’s medium, or a suitable equivalent, is the
6.2 Containers—Flasks, bottles, or test tubes suitable for
recommended medium for the growth of algae.
shaking shall be sterilized prior to use. 7.4 Dilution Water Blanks—Sterile, 99 or 9-mL phosphate
6.3 Colony Counters—Manual, such as Quebec, Buck, or
buffered saline or magnesium chloride dilution blanks are
Wolffhuegel, or a proven colony image analyzer (electronic/ convenient for diluting test samples for viable counts. Buffer
scanner type) are suitable for counting plates after incubation.
strength and salinity can be adjusted to mimic experimental or
A hand tally or automatic recording device on the manual field conditions.
counter is desirable.
7.4.1 Phosphate Buffered Dilution Water Blanks.
6.4 Spiral Plater.
7.4.1.1 Phosphate Buffer Solution, Stock—Dissolve 34.0 g
6.5 Constant Temperature Shaker—A reliable constant-
of potassium dihydrogen phosphate (KH PO ) in 500 mL of
2 4
temperature shaker 62°C (water bath or incubator shaker)
water. Adjust pH to 7.2 6 0.2 with NaOH solution (40 g/L) and
shall be used to provide mixing and aeration and to maintain
bring to 1000 mL with water. Sterilize by filtration or auto-
temperature during the contact period at a setting within the
clave.
temperature range selected in 10.2.
7.4.1.2 Phosphate Buffered Saline Dilution Water—Add
6.6 Petri Dishes, sterile, 100 by 15-mm plastic or borosili-
1.25 mL of stock phosphate buffer solution and 8.75 g of NaCl
cate glass.
to a volumetric flask, fill with reagent water to the 1000-mL
6.7 Pipettes—Standard pipettes, sterile, with appropriate
mark, and mix. Final pH should be 7.2 6 0.2. Dispense in
calibrations, or other suitable delivery systems, such as mi-
amount that will provide 99 6 2mL or9 6 1 mL after
cropipetters, can be employed.
sterilization into screw-cap dilution bottles or tubes. Sterilize
6.8 Sterilizers—Pressurized steam sterilizer (for media, immediately.
containers, and so forth), hot air oven (180 6 2°C for 2 h) for
7.4.2 Phosphate Buffered Magnesium Chloride Dilution
containers, and filter apparatus for filter sterilization (dispos- Water—Add 1.25 mL of stock phosphate buffer solution and
able filter units, 250 mL, 0.22-μm pore size).
5.0 mL of magnesium chloride solution (81.1 g MgCl 6
6.9 Stirrer—A stirrer is required to mix the cooling water H O/L, reagent grade water) to 1000 mL of water. Adjust pH
sample while it is being dispensed into test containers. This can
to 7.2 6 0.2. Dispense in amount that will provide 99 6 2mL
be a magnetic stirrer, a propeller-type stirrer, or any other or 9 6 1 mL after sterilization into screw-cap dilution bottles
suitable device.
or tubes. Sterilize immediately.
6.10 Volumetric Flasks, 100 mL, are convenient for prepar-
7.5 Cooling Tower Water Sample:
ing microbicide stock solutions. Smaller volume flasks may be
7.5.1 The cooling tower water sample shall be collected in
used where appropriate.
a sterile container (1-gal or 2.2-L plastic bottles are conve-
6.11 Blender—A blender, stomacher, sonic bath, or vortex
nient). The temperature and pH should be determined at the
mixer, may be necessary to homogenize the microbial deposit
time of sample collection. The presence of additives in the
before mixing it with the cooling water.
cooling tower water may affect the efficacy of the microbicides,
6.12 Microscope, provides a magnification of 400 to 10003
therefore, a history of the samples should be obtained or
and is complete with a suitable light source. Phase contrast or
analysis of the additives should be conducted. The samples
dark-field capability is desirable.
7. Reagents and Materials
Starr, R. C., and Zeikus, J. A., “The Culture Collection of Algae at the
7.1 Purity of Reagents—The principal reagents used are the University of Texas at Austin,” Journal of Psychology, Vol 23(5): pp. 1–47, 1987.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 645
shall not be exposed to temperature extremes during transit. If 25.0, and 50.0-mg/L test concentrations, by addition of 0.2,
a variation of 1.0 pH unit exists between the time of sampling 0.5, and 1.0 mL, respectively, per 100 mL of sample. The
and testing, the sample should be discarded. The test procedure second stock is used to make the more dilute test concentra-
should be initiated within 24 h after collection. tions. Using this method, the volume of microbicide stock
7.5.2 Deposits of microbial composition should be collected solution added will not exceed 1 % of the total volume of the
in sterile containers from any affected areas of the cooling cooling water aliquots. This relative volume is an important
tower, such as the distribution deck, slats, or sump area. The consideration, particularly when solvents other than water are
deposit samples should be transported with the water sample used to make the microbicide stock solutions. If an acceptable
following the same precautions. Upon receipt at the laboratory, reduction in microbial numbers will not be achieved with these
microscopic examination of the deposits should be conducted concentrations, the investigator must make the appropriate
to confirm that it is microbiological in nature. If testing for adjustments in the microbicide stock solutions and in the
algicide or fungicide activity, or both, the sample must contain selected test concentrations in order to achieve this end.
algae or fungi, or both. Microbicide test stock solutions should be prepared no more
than 3 h before the test.
8. Preparation of the Test Samples
9.1.1 The investigator will determine the minimal concen-
tration for efficacy based upon customer/laboratory registration
8.1 The cooling water sample may be used as received or
needs.
inoculated with known microorganisms. If only the water is
9.2 The initial microbicide stock solution is prepared by
used as a substrate and known microorganisms will be added
weighing the microbicide on an analytical balance, transferring
as inoculum, then the water should be filter-sterilized prior to
it to a volumetric flask, and bringing it to correct volume.
the addition of microorganisms. If a biofilm sample or micro-
Aluminum weighing dishes are not recommended because of
biological deposit is available, it may be used as the inoculum
the reactive nature of aluminum.
in filter-sterilized cooling water or synthetic cooling water. A
homogenate should be prepared with the biofilm deposit not
10. Addition of Microbicide to Test Samples
more than 10 % of the total weight of the samples.
8.2 The cooling water sample should be placed on a stirrer
10.1 It is necessary to stagger the starting times by adding
and mixed continuously. Transfer 100 6 2 mL (or 100 6 2g) the microbicide stock solution to the test aliquots at timed
to sterile flasks or bottles. Prepare at least duplicate flasks or
intervals. Intervals of 1.5 to 2 min are convenient, but the time
bottles for each microbicide concentration to be tested. In selected will depend on the speed of the investigator. The test
addition, prepare duplicate controls to which no microbicide
flasks should be plated in the same order in which the
will be added. If a solvent other than water is used to make the microbicide was added.
microbicide stock solutions, also include solvent control
10.2 Since different microbicides vary in their mode of
bottles that contain as much of the solvent as is added to the action, the exposure time should be consistent with the
microbic
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