ASTM E645-18

Standard Practice for Evaluation of Microbicides Used in Cooling Water Systems

SIGNIFICANCE AND USE
5.1 This practice determines potentially effective microbicides for use in cooling water systems using cooling water and deposits/biofilm obtained from the field. The addition of deposits/biofilms addresses the need to include the major source of microorganisms in cooling water systems. Even with this addition, laboratory results may not be totally predictive of microbicidal effectiveness in the field. This is because conditions in the field affecting microbicide effectiveness are difficult to mimic in the laboratory. These conditions that affect microbicide efficacy include blow-down rate, addition of makeup water, water hardness, hydrocarbon leaks, pH, sediment loading, dissolved solids, microbes in slime (biofilms), and deposits (salts, iron minerals, organics, and so forth) on surfaces. An additional factor is the difficulty in enumerating all microbes in the water due to the lack of adequate recovery media. Guidelines that address formation of and testing for surface-attached microbes (biofilms) may be found in Guide E1427, while a guideline for unconventional measurement of microbes is found in Guide E1326.
SCOPE
1.1 This practice 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 (1) microbes from cooling water, (2) microbes in microbiological deposits (biofilms) from operating cooling systems, or (3) microorganisms known to contaminate cooling water systems, or a combination thereof. This practice should be performed by individuals familiar with microbiological techniques.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 30-Sep-2018
• ICS: 65.100.99 - Other pesticides and agrochemicals
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Replaces: ASTM E645-13 - Standard Practice for Evaluation of Microbicides Used in Cooling Water Systems
• Effective Date: 01-Oct-2018
• Refers: ASTM D4412-19(2024) - Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
• Effective Date: 01-Apr-2024
• Refers: ASTM D4012-23a - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
• Effective Date: 01-Nov-2023
• Refers: ASTM D3731-20 - Standard Practices for Measurement of Chlorophyll Content of Algae in Surface Waters
• Effective Date: 01-Jun-2020
• Refers: ASTM D4412-19 - Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
• Effective Date: 01-Dec-2019
• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM E1326-15 - Standard Guide for Evaluating Non-culture Microbiological Tests Used for Enumerating Bacteria
• Effective Date: 01-May-2015
• Refers: ASTM E1326-13 - Standard Guide for Evaluating Nonconventional Microbiological Tests Used for Enumerating Bacteria
• Effective Date: 01-Oct-2013
• Refers: ASTM D3731-87(2012) - Standard Practices for Measurement of Chlorophyll Content of Algae in Surface Waters
• Effective Date: 01-Sep-2012
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM D4412-84(2009) - Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
• Effective Date: 01-May-2009
• Refers: ASTM D4012-81(2009) - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
• Effective Date: 01-May-2009
• Refers: ASTM E1326-08 - Standard Guide for Evaluating Nonconventional Microbiological Tests Used for Enumerating Bacteria
• Effective Date: 01-Oct-2008
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008

Overview

ASTM E645-18 is a standard practice developed by ASTM International for the evaluation of microbicides utilized in cooling water systems. Microbicides, including algicides, bactericides, and fungicides, play a crucial role in controlling microbial growth such as bacteria, algae, and fungi in industrial cooling water environments. This document provides a standardized approach for testing the effectiveness of these agents using water and biofilm samples collected directly from the field, simulating real operational conditions as closely as possible.

The practice addresses the inherent challenges in predicting real-world performance because laboratory simulations cannot perfectly reproduce all field conditions, such as variable water chemistry, biofilm formation, and operational parameters. ASTM E645-18 helps ensure that microbicide products selected for cooling towers and other water-based heat exchange systems demonstrate effective microbial control under varied and challenging environments.

Key Topics

• Microbicide Efficacy Evaluation: Outlines methods for testing the effectiveness of microbicides against field-derived waterborne microorganisms and biofilms.
• Sample Preparation: Guidance on collecting, storing, and preparing cooling water and deposits/biofilms for testing, ensuring representative microbial populations.
• Test Procedures: Recommendations for laboratory procedures such as mixing, incubation times, and microbial enumeration using methods like plate counting and ATP measurement.
• Interpretation of Results: Instructions for determining log reduction and percent kill, with criteria for meaningful microbial reduction and test validity.
• Field-Laboratory Correlation: Discussion of the limitations when extrapolating laboratory results to real-world settings due to factors like water hardness, blow-down, pH variations, and organic/inorganic deposits.
• Safety and Compliance: Emphasizes user responsibility for safety, health, and environmental practices and regulatory compliance.

Applications

ASTM E645-18 finds practical use in:

• Cooling Tower Maintenance: Supporting the selection of effective biocides for industrial and commercial cooling towers, recirculating systems, and once-through cooling systems.
• Product Development and Registration: Used by microbicide manufacturers to demonstrate product performance and satisfy regulatory or customer requirements.
• Microbial Contamination Control: Assisting facility operators, water treatment professionals, and consultants in controlling biofouling and microbiologically influenced corrosion (MIC) in cooling water systems.
• Testing Against Real-World Samples: Providing guidelines for using actual field samples, including biofilms and deposits, ensuring that microbicides are tested under relevant conditions.

Employing this standard helps optimize biocide dosing, minimize microbial-related equipment failures, and ensure regulatory compliance for water system operation.

Related Standards

Users of ASTM E645-18 may also refer to the following related standards for comprehensive microbial testing in water systems:

• ASTM D3731: Practices for Measurement of Chlorophyll Content of Algae in Surface Waters
• ASTM D4012: Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
• ASTM D4412: Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
• ASTM E1054: Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• ASTM E1326: Guide for Evaluating Non-culture Microbiological Tests
• ASTM E1427: Guide for Selecting Test Methods to Determine the Effectiveness of Antimicrobial Agents and Other Chemicals for the Prevention, Inactivation, and Removal of Biofilm
• ASTM E2756: Terminology Relating to Antimicrobial and Antiviral Agents

Adopting ASTM E645-18 alongside these related microbiological testing standards ensures robust, field-relevant, and compliant microbicide evaluation for the effective management of microbial contamination in cooling water systems.

Keywords: ASTM E645-18, cooling water systems, microbicides, biocide efficacy, microbial control, cooling towers, biofilms, standard practice, water treatment, industrial water systems.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E645 − 18
Standard Practice for
Evaluation of Microbicides Used in Cooling Water Systems
This standard is issued under the fixed designation E645; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope* and Water-Formed Deposits
E1054Test Methods for Evaluation of Inactivators of Anti-
1.1 This practice outlines a procedure for evaluating the
microbial Agents
efficacy of microbicides (algicides, bactericides, and fungi-
E1326Guide for Evaluating Non-culture Microbiological
cides) that will be used for controlling microbial growth in
Tests
cooling water systems. The microbicides will be evaluated
E1427Guide for Selecting Test Methods to Determine the
using simulated or real cooling tower water against (1)
Effectiveness of Antimicrobial Agents and Other Chemi-
microbes from cooling water, (2) microbes in microbiological
cals for the Prevention, Inactivation and Removal of
deposits (biofilms) from operating cooling systems, or (3)
Biofilm (Withdrawn 2009)
microorganisms known to contaminate cooling water systems,
E2756Terminology Relating toAntimicrobial andAntiviral
oracombinationthereof.Thispracticeshouldbeperformedby
Agents
individuals familiar with microbiological techniques.
1.2 The values stated in SI units are to be regarded as
3. Terminology
standard. No other units of measurement are included in this
3.1 For definitions of terms used in this practice, see
standard.
Terminology E2756.
1.3 This standard does not purport to address all of the
3.2 Definitions of Terms Specific to This Standard:
safety concerns, if any, associated with its use. It is the
3.2.1 algicide, n—a chemical agent that kills algae; unicel-
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter- lular or filamentous chlorophyll-containing plants.
mine the applicability of regulatory limitations prior to use.
3.2.2 bactericide, n—a physical or chemical agent that kills
1.4 This international standard was developed in accor-
bacteria, but not necessarily bacterial spores.
dance with internationally recognized principles on standard-
3.2.3 biofilm, n—adynamic,self-organizedaccumulationof
ization established in the Decision on Principles for the
microorganisms and environmental by-products immobilized
Development of International Standards, Guides and Recom-
on a substrate and embedded in an organic polymer matrix.
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
3.2.4 cooling system, n—equipmentandcoolantusedforthe
removal of heat from processes, equipment, or both.
2. Referenced Documents
3.2.4.1 Discussion—The most common medium used for
removal or transfer of heat is water.The heated water then can
2.1 ASTM Standards:
be discharged into a receiving body (once through cooling
D3731PracticesforMeasurementofChlorophyllContentof
system) or it can be cooled and reused (recirculating cooling
Algae in Surface Waters
system).
D4012TestMethodforAdenosineTriphosphate(ATP)Con-
tent of Microorganisms in Water
3.2.5 cooling tower, n—a structure used to dissipate heat in
D4412TestMethodsforSulfate-ReducingBacteriainWater
open recirculating cooling systems.
3.2.6 cooling water, n—any water-based solution that ab-
sorbs and transfers heat in a heat exchange system.
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
3.2.7 fungicides, n—a physical or chemical agent that kills
Subcommittee E35.15 on Antimicrobial Agents.
fungi;thatis,vegetativemyceliaand/orbuddingyeastsinclud-
Current edition approved Oct. 1, 2018. Published October 2018. Originally
ing spores and/or conidia.
approved in 1978. Last previous edition approved in 2013 as E645–13. DOI:
10.1520/E0645-18.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on The last approved version of this historical standard is referenced on
the ASTM website. www.astm.org.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E645 − 18
3.2.8 microbial biofouling, n—the unwanted accumulation during the contact period at a setting within the temperature
of bacterial, fungal, or algal cells, or any combination thereof range selected in 10.2.
and their products on surfaces.
6.6 Petri Dishes, sterile, 100 by 15-mm plastic or borosili-
3.2.8.1 Discussion—Often this accumulation is accompa-
cate glass.
nied by deposition of organic and inorganic material.
6.7 Pipettes—standard pipettes, sterile, with appropriate
3.2.9 microbicides, n—a physical or chemical agent that
calibrations, or other suitable delivery systems, such micropi-
kills microorganisms.
petters.
4. Summary of Practice
6.8 Sterilizers—pressurized steam sterilizer (for media,
4.1 Microbicides are evaluated against microbes under con-
containers, and so forth), hot air oven for containers, and filter
ditions simulating a cooling water system. Microbicides at
apparatusforfiltersterilization(disposablefilterunits,250mL,
concentrations that are expected to control the microbes are
0.22-µm pore size).
addedtocoolingwater.Atselectedtimeperiods,thenumberof
6.9 Stirrer—required to mix the cooling water sample while
microbes or measurable component of the microbes are deter-
it is being dispensed into test containers. This can be a
mined and compared to values at the start of the experiment.
magnetic stirrer, a propeller-type stirrer, or any other suitable
Bacteria (aerobic and anaerobic), fungi, and algae may be
device.
detectedbyanumberofmethods,suchasplatecounting,Most
Probable Number (MPN), chlorophyll content, adenosine-5ʹ-
6.10 Volumetric Flasks, 100 mL, are convenient for prepar-
triphosphate (ATP). The investigator will determine the range
ing microbicide stock solutions. Smaller volume flasks may be
of microbicide concentration for acceptable efficacy based
used where appropriate.
upon laboratory testing that may be used to satisfy registration
6.11 Blender—a blender, stomacher, sonic bath, or vortex
or customer needs.
mixer to homogenize the microbial deposit before mixing it
with the cooling water.
5. Significance and Use
5.1 This practice determines potentially effective microbi-
6.12 Microscope, providing a magnification range of 400 to
cides for use in cooling water systems using cooling water and
1000× with a suitable light source. Phase contrast or dark-field
deposits/biofilm obtained from the field. The addition of
capability may be necessary.
deposits/biofilms addresses the need to include the major
6.13 Filter apparatus, with 0.2 µm filter.
source of microorganisms in cooling water systems. Even with
thisaddition,laboratoryresultsmaynotbetotallypredictiveof
7. Reagents and Materials
microbicidal effectiveness in the field. This is because condi-
tions in the field affecting microbicide effectiveness are diffi-
7.1 Purity of Reagents—Theprincipalreagentusediswater,
cult to mimic in the laboratory. These conditions that affect
but other solvents may be necessary in preparing the microbi-
microbicide efficacy include blow-down rate, addition of
cide stock solutions. Reagent grade organic solvents are
makeup water, water hardness, hydrocarbon leaks, pH, sedi-
normally used if water is not a suitable diluent for dissolving a
ment loading, dissolved solids, microbes in slime (biofilms),
microbicide. If a solvent is used, an additional control must be
and deposits (salts, iron minerals, organics, and so forth) on
performed that has solvent without any microbicide added to
surfaces. An additional factor is the difficulty in enumerating
the cooling water sample. This is used to demonstrate that the
all microbes in the water due to the lack of adequate recovery
solvent has no appreciable effect on the test results.
media. Guidelines that address formation of and testing for
7.2 Purity of Water—All reference to water as a diluent or
surface-attached microbes (biofilms) may be found in Guide
reagent shall mean distilled water or water of equal purity,
E1427, while a guideline for unconventional measurement of
unless otherwise noted.
microbes is found in Guide E1326.
7.3 Culture Media:
6. Apparatus
7.3.1 A general bacterial agar medium, such as glucose
6.1 Balance—a calibrated analytical balance sensitive to
extract agar, tryptic soy agar, R2Aagar, or dry film is used for
0.1mg to weigh the candidate microbicide for preparation of
conductingbacterialcountsontestsamples.Othermedia,such
stock solutions.
as selective or differential types (that is, for the quantification
ofsulfate-reducingbacteria,TestMethodsD4412)maybeused
6.2 Containers—flasks, bottles, or test tubes suitable for
for detecting desired bacteria. MPN or ATP (Test Method
shaking shall be sterile for use.
D4012) measurement may also be used to quantify the biobur-
6.3 Colony Counters—manual, such as Quebec, Buck, or
dens (Guide E1326). Once a specific agar medium or other
Wolffhuegel, or a proven colony image analyzer (electronic/
method of measurement is chosen, it must be used throughout
scanner type) are suitable for counting plates after incubation.
this procedure.
6.4 Spiral Plater (alternative).
7.3.2 Ageneral fungal medium, such as an inhibitory mold
6.5 Constant Temperature Shaker—a reliable constant- agar or Sabouraud dextrose agar, is used for conducting fungal
temperature shaker 62°C (water bath or incubator shaker) to countsonthesamples.Thismediummustbeabletoinhibitthe
provide mixing and aeration and to maintain temperature growth of bacteria.
E645 − 18
4 5
7.3.3 Bristol’s medium, or a suitable equivalent, is the only as a substrate and known microorganisms will be added
recommended medium for the growth of algae. as inoculum, the water should be filter-sterilized (using a
0.2µmfiltersystem)priortotheadditionofmicroorganisms.If
7.4 Dilution Water Blanks—Sterile, 99 or 9-mL phosphate
abiofilmsampleormicrobiologicaldepositisavailable,itmay
buffered saline or phosphate buffered magnesium chloride
be used as the inoculum in either filtered or non-filtered
dilution blanks are convenient for diluting test samples for
sterilized cooling water. The biolfilm or slime must be
viable counts. Buffer strength and salinity can be adjusted to
homogenized/disaggregatedsothatnoclumpsarepresent.This
mimic experimental or field conditions.
can be accomplished by vortexing, sonicating, or any other
7.4.1 Phosphate Buffered Dilution Water Blanks.
method that disperses the clumps. No more than 10 % of the
7.4.1.1 Phosphate Buffer Solution, Stock—Dissolve 34.0 g
total weight (w/v) of the samples should be biofilm or deposit.
of potassium dihydrogen phosphate (KH PO ) in 500 mL of
2 4
A synthetic cooling water may also be used as the sample
water.AdjustpHto7.2 60.2withNaOHsolution(40g/L)and
water.
bring to 1000 mL with water. Sterilize by filtration or auto-
clave. 8.2 Place the cooling water sample on a stirrer and mix
7.4.1.2 Phosphate Buffered Saline Dilution Water—Add continuously. Transfer 100 6 2 mL (or 100 6 2 g) to sterile
1.25 mLof stock phosphate buffer solution and 8.75 g of NaCl flasks or bottles. Prepare at least duplicate flasks or bottles for
to a volumetric flask, fill with reagent water to the 1000-mL each microbicide concentration to be tested. In addition,
mark, and mix. Final pH should be 7.2 6 0.2. Dispense in prepare duplicate controls to which no microbicide will be
amount that will provide 99 62mLor9 6 1 mL after added. If a solvent other than water is used to make the
sterilization into screw-cap dilution bottles or tubes. Sterilize microbicide stock solutions, also include solvent control
immediately. bottles that contain as much of the solvent as is added to the
7.4.2 Phosphate Buffered Magnesium Chloride Dilution microbicide test containers (see 8.1). The 100-mL water
Water—Add 1.25 mL of stock phosphate buffer solution and volume is a standard volume used in all previously published
5.0 mL of magnesium chloride solution (81.1 g MgCl ·6 evaluation of cooling water microbicides. However, other
H O/L, reagent grade water) to 1000 mL of water. Adjust pH volumes of cooling water may be used in this test.
to7.2 60.2.Dispenseinamountsthatwillprovide99 62mL
8.3 After the test aliquots have been transferred to flasks,
or 9 6 1 mL after sterilization into screw-cap dilution bottles
determine the viable count of
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E645 − 13 E645 − 18
Standard Practice for
Evaluation of Microbicides Used in Cooling Water Systems
This standard is issued under the fixed designation E645; 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 Scope*
1.1 This practice 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 (1) microbes from cooling water, (2) microbes in microbiological deposits (biofilms) from operating
cooling systems, or (3) microorganisms known to contaminate cooling water systems, or a combination thereof. This practice
should be performed by individuals familiar with microbiological techniques.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.4 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D3731 Practices for Measurement of Chlorophyll Content of Algae in Surface Waters
D4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
D4412 Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents
E1326 Guide for Evaluating Non-culture Microbiological Tests
E1427 Guide for Selecting Test Methods to Determine the Effectiveness of Antimicrobial Agents and Other Chemicals for the
Prevention, Inactivation and Removal of Biofilm (Withdrawn 2009)
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
3. Terminology
3.1 For definitions of terms used in this practice, see Terminology E2756.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 algicide, n—a chemical agent that kills algae; unicellular or filamentous chlorophyll-containing plants.
3.2.2 bactericide, n—a physical or chemical agent that kills bacteria, but not necessarily bacterial spores.
3.2.3 biofilm, n—a dynamic, self-organized accumulation of microorganisms and environmental by-products immobilized on a
substrate and embedded in an organic polymer matrix.
3.2.4 cooling system, n—equipment and coolant used for the removal of heat from processes, equipment, or both.
3.2.4.1 Discussion—
This practice is under the jurisdiction of ASTM 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, 2013Oct. 1, 2018. Published May 2013October 2018. Originally approved in 1978. Last previous edition approved in 20072013 as
E645 – 07.E645 – 13. DOI: 10.1520/E0645-13.10.1520/E0645-18.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E645 − 18
The most common medium used for removal or transfer of heat is water. The heated water then can be discharged into a receiving
body (once through cooling system) or it can be cooled and reused (recirculating cooling system).
3.2.5 cooling tower, n—a structure used to dissipate heat in open recirculating cooling systems.
3.2.6 cooling water, n—any water-based solution that absorbs and transfers heat in a heat exchange system.
3.2.7 fungicides, n—a physical or chemical agent that kills fungi; that is, vegetative mycelia and/or budding yeasts including
spores and/or conidia.
3.2.8 microbial biofouling, n—the unwanted accumulation of bacterial, fungal, or algal cells, or any combination thereof and
their products on surfaces.
3.2.8.1 Discussion—
Often this accumulation is accompanied by deposition of organic and inorganic material.
3.2.9 microbicides, n—a physical or chemical agent that kills microorganisms.
4. Summary of Practice
4.1 Microbicides are evaluated against microbes under conditions simulating a cooling water system. Microbicides at
concentrations that are expected to control the microbes are added to cooling water. At selected time periods, the number of
microbes or measurable component of the microbes are determined and compared to values at the start of the experiment. Bacteria
(aerobic and anaerobic), fungi, and algae may be detected by a number of methods, such as plate counting, Most Probable Number
(MPN), chlorophyll content, adenosine-5ʹ-triphosphate (ATP). The investigator will determine the range of microbicide
concentration for acceptable efficacy based upon laboratory testing that may be used to satisfy registration or customer needs.
5. Significance and Use
5.1 This practice determines potentially effective microbicides for use in cooling water systems using cooling water and
deposits/biofilm obtained from the field. The addition of deposits/biofilms addresses the need to include the major source of
microorganisms in cooling water systems. Even with this addition, laboratory results may not be totally predictive of microbicidal
effectiveness in the field. This is because conditions in the field affecting microbicide effectiveness are difficult to mimic in the
laboratory. These conditions that affect microbicide efficacy include blow-down rate, addition of makeup water, water hardness,
hydrocarbon leaks, pH, sediment loading, dissolved solids, microbes in slime (biofilms), and deposits (salts, iron minerals,
organics, and so forth) on surfaces. An additional factor is the difficulty in enumerating all microbes in the water due to the lack
of adequate recovery media. Guidelines that address formation of and testing for surface-attached microbes (biofilms) may be
found in Guide E1427, while a guideline for unconventional measurement of microbes is found in Guide E1326.
6. Apparatus
6.1 Balance—a calibrated analytical balance sensitive to 0.1 mg to weigh the candidate microbicide for preparation of stock
solutions.
6.2 Containers—flasks, bottles, or test tubes suitable for shaking shall be sterile for use.
6.3 Colony Counters—manual, such as Quebec, Buck, or Wolffhuegel, or a proven colony image analyzer (electronic/scanner
type) are suitable for counting plates after incubation.
6.4 Spiral Plater (alternative).
6.5 Constant Temperature Shaker—a reliable constant-temperature shaker 62°C62 °C (water bath or incubator shaker) to
provide mixing and aeration and to maintain temperature during the contact period at a setting within the temperature range
selected in 10.2.
6.6 Petri Dishes, sterile, 100 by 15-mm plastic or borosilicate glass.
6.7 Pipettes—standard pipettes, sterile, with appropriate calibrations, or other suitable delivery systems, such micropipetters.
6.8 Sterilizers—pressurized steam sterilizer (for media, containers, and so forth), hot air oven for containers, and filter apparatus
for filter sterilization (disposable filter units, 250 mL, 0.22-μm pore size).
6.9 Stirrer—required to mix the cooling water sample while it is being dispensed into test containers. This can be a magnetic
stirrer, a propeller-type stirrer, or any other suitable device.
6.10 Volumetric Flasks, 100 mL, are convenient for preparing microbicide stock solutions. Smaller volume flasks may be used
where appropriate.
6.11 Blender—a blender, stomacher, sonic bath, or vortex mixer to homogenize the microbial deposit before mixing it with the
cooling water.
E645 − 18
6.12 Microscope, providing a magnification range of 400 to 1000× with a suitable light source. Phase contrast or dark-field
capability may be necessary.
6.13 Filter apparatus, with 0.2 μm filter.
7. Reagents and Materials
7.1 Purity of Reagents—The principal reagent used is water, but other solvents may be necessary in preparing the microbicide
stock solutions. Reagent grade organic solvents are normally used if water is not a suitable diluent for dissolving a microbicide.
If a solvent is used, an additional control must be performed that has solvent without any microbicide added to the cooling water
sample. This is used to demonstrate that the solvent has no appreciable effect on the test results.
7.2 Purity of Water—All reference to water as a diluent or reagent shall mean distilled water or water of equal purity, unless
otherwise noted.
7.3 Culture Media:
7.3.1 A general bacterial agar medium, such as glucose extract agar, tryptic soy agar, R2A agar, or dry film is used for
conducting bacterial counts on test samples. Other media, such as selective or differential types (that is, for the quantification of
sulfate-reducing bacteria, Test Methods D4412) may be used for detecting desired bacteria. MPN or ATP (Test Method D4012)
measurement may also be used to quantify the bacteriabioburdens (Guide E1326). Once a specific agar medium or other method
of measurement is chosen, it must be used throughout this procedure.
7.3.2 A general fungal medium, such as an inhibitory mold agar or Sabouraud dextrose agar, is used for conducting fungal
counts on the samples. This medium must be able to inhibit the growth of bacteria.
7.3.3 Bristol’s medium, or a suitable equivalent, is the recommended medium for the growth of algae.
7.4 Dilution Water Blanks—Sterile, 99 or 9-mL phosphate buffered saline or phosphate buffered magnesium chloride dilution
blanks are convenient for diluting test samples for viable counts. Buffer strength and salinity can be adjusted to mimic experimental
or field conditions.
7.4.1 Phosphate Buffered Dilution Water Blanks.
7.4.1.1 Phosphate Buffer Solution, Stock—Dissolve 34.0 g of potassium dihydrogen phosphate (KH PO ) in 500 mL of water.
2 4
Adjust pH to 7.2 6 0.2 with NaOH solution (40 g/L) and bring to 1000 mL with water. Sterilize by filtration or autoclave.
7.4.1.2 Phosphate Buffered Saline Dilution Water—Add 1.25 mL of stock phosphate buffer solution and 8.75 g of NaCl to a
volumetric flask, fill with reagent water to the 1000-mL mark, and mix. Final pH should be 7.2 6 0.2. Dispense in amount that
will provide 99 6 2 mL or 9 6 1 mL after sterilization into screw-cap dilution bottles or tubes. Sterilize immediately.
7.4.2 Phosphate Buffered Magnesium Chloride Dilution Water—Add 1.25 mL of stock phosphate buffer solution and 5.0 mL
of magnesium chloride solution (81.1 g MgCl · 6 H O/L, reagent grade water) to 1000 mL of water. Adjust pH to 7.2 6 0.2.
2 2
Dispense in amounts that will provide 99 6 2 mL or 9 6 1 mL after sterilization into screw-cap dilution bottles or tubes. Sterilize
immediately.
7.5 Cooling Water Sample:
7.5.1 The cooling water sample will be collected in a sterile container (1-gal or 2.2-L plastic bottles are convenient). The
temperature and pH should be determined at the time of sample collection. The presence of additives in the cooling tower water
may affect the effectiveness of the microbicides, therefore, a history of the samples should be obtained or analysis of the water for
additives should be conducted. Stop biocide addition at least 4 h before collection of samples, or an appropriate biocide inactivator
must be added to the sample. Do not expose samples to temperature extremes during transit. If a variation of 1.0 pH unit exists
between the time of sampling and testing, the sample should be discarded. The test procedure should be initiated within 24 h after
collection. Samples received from the field must be refrigerated (4 6 2ºC).(4 6 2 °C).
7.5.2 Collect deposits of microbial composition in sterile containers from any affected areas of the cooling tower, such as the
distribution deck, slats, or sump area. Transport the deposit samples with the water sample following the same precautions. Upon
receipt at the laboratory, conduct microscopic examination of the deposits to confirm that they are microbiological in nature. If
testing for algicidal or fungicidal activity, or both, the sample must contain algae or fungi, or both.
8. Preparation of the Test Samples
8.1 The cooling water s
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