ASTM E1259-23

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: E1259 − 18 E1259 − 23
Standard Practice for
Evaluation of Antimicrobials in Liquid Fuels Boiling Below
390 °C
This standard is issued under the fixed designation E1259; 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*
1.1 This practice is designed to evaluate antimicrobial agents for the prevention of microbially influenced deterioration of liquid
fuels (as defined by Specification D396, D910, D975, D1655, D2069, D2880, D3699, D4814, D6227, D6751, and D7467), system
deterioration, or both.
1.2 Knowledge of microbiological techniques is required for these procedures.
1.3 It is the responsibility of the investigator to determine whether Good Laboratory Practice (GLP) is required and to follow them
where appropriate (40 CFR, 160), or as revised.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 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.
1.6 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:
D396 Specification for Fuel Oils
D910 Specification for Leaded Aviation Gasolines
D975 Specification for Diesel Fuel
D1655 Specification for Aviation Turbine Fuels
D2069 Specification for Marine Fuels (Withdrawn 2003)
D2880 Specification for Gas Turbine Fuel Oils
D3699 Specification for Kerosine
D4814 Specification for Automotive Spark-Ignition Engine Fuel
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 Oct. 1, 2018June 1, 2023. Published October 2018June 2023. Originally approved in 1988. Last previous edition approved in 20162018 as
E1259 – 16.E1259 – 18. DOI: 10.1520/E1259-18.10.1520/E1259-23.
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 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
E1259 − 23
D5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
D6227 Specification for Unleaded Aviation Gasoline Containing a Non-hydrocarbon Component
D6293 Test Method for Oxygenates and Paraffin, Olefin, Naphthene, Aromatic(O-PONA) Hydrocarbon Types in Low-Olefin
Spark Ignition Engine Fuels by Gas Chromatography (Withdrawn 2009)
D6469 Guide for Microbial Contamination in Fuels and Fuel Systems
D6729 Test Method for Determination of Individual Components in Spark Ignition Engine Fuels by 100 Metre Capillary High
Resolution Gas Chromatography
D6733 Test Method for Determination of Individual Components in Spark Ignition Engine Fuels by 50-Metre Capillary High
Resolution Gas Chromatography
D6751 Specification for Biodiesel Fuel Blendstock (B100) for Middle Distillate Fuels
D6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
D7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel
Associated Water
D7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological
Testing
D7467 Specification for Diesel Fuel Oil, Biodiesel Blend (B6 to B20)
D7687 Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel and Fuel-associated Water With Sample
Concentration by Filtration
D7978 Test Method for Determination of the Viable Aerobic Microbial Content of Fuels and Associated Water—Thixotropic Gel
Culture Method
D8412 Guide for Quantification of Microbial Contamination in Liquid Fuels and Fuel-Associated Water by Quantitative
Polymerase Chain Reaction (qPCR)
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E1259 Practice for Evaluation of Antimicrobials in Liquid Fuels Boiling Below 390 °C
E1326 Guide for Evaluating Non-culture Microbiological Tests
2.2 NACE Standard:
TM0172 Determining Corrosive Properties of Cargoes in Petroleum Product Pipelines
2.3 Federal Standards:
40 CFR Part 79 Fuels and Fuel Additives Registration Regulations
40 CFR Part 152 Pesticide Registration and Classification Procedures
3. Terminology
3.1 Definitions of Terms Specific to This Standard:
3.1.1 antimicrobial, n—see biocide.
3.1.2 biocide, n—a physical or chemical agent that kills living organisms.
3.1.2.1 Discussion—
Biocides are further classified as bactericides (kill bacteria), fungicides (kill fungi), and microbicides (kill both bacterial and fungi).
They are also referred to as antimicrobials.
3.1.3 microbially-influenced deterioration, n—decomposition /degradation of material (fuel) or making unsuitable for use, as a
result of metabolic activity or the presence of microbes.
3.1.4 microbicide, n—see biocide.
3.1.5 microcosm, n—a miniature system used to model larger systems.
3.1.5.1 Discussion—
It is generally impractical to evaluate microbicide performance in large fuel storage system capacities (> 24 000 m ), consequently
small volume (1.0 to 208 L capacity) microcosms are used as model systems.
Item No. 21204, available from NACE International (NACE), 1440 South Creek Dr., Houston, TX 77084-4906, http://www.nace.org.
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.
E1259 − 23
4. Summary of Practice
4.1 This practice is conducted on a fuel representative of the grade to be treated, and determines the antimicrobial efficacy under
well-defined conditions that may include specific inocula or an uncharacterized inoculum from a microbially contaminated fuel
system.
4.1.1 Water/fuel ratios and containment time are also defined. This practice allows for impact of fuel/water partitioning and time,
on the antimicrobial agent, as well as the effect of continual rechallenge.
4.1.2 At each sampling time interval, treated and untreated aliquots are checked for the treated population survival. survival,
proliferation, or both. Microbiological testing is coupled with gross observations of each system for biofilm formation and
interfacial growth.
4.1.3 The size of the test system, total volume of fluid, fuel to bottom-water ratio and test duration ratio, test duration, and test
system incubation temperature may vary depending on the specific objectives of the test.
4.1.4 Before beginning any test plan intended to meet performance testing compliance requirements, confirm that the cognizant
authority accepts the test protocol.
5. Significance and Use
5.1 Guide D6469 details the types of problems associated with uncontrolled microbial growth in fuels and fuel systems. Treatment
with effective antimicrobial agents is one element of contamination control strategy.
5.2 The procedure should be used to evaluate the relative efficacy of microbicides in liquid fuels boiling below 390 °C. The effect
of environmental conditions, such as a variety of fuel additives, metal surfaces, and climatology, are variables that can be included
in specific tests using this protocol.
5.3 This practice addresses product performance issues only. Regulatory Agencies restrict and control the use of both pesticides
(in the U.S.: 40 CFR 152) and fuel additives (40 CFR 79). Regardless of performance in this method, antimicrobials must only
be used in compliance with applicable regulations. Specific industries, for example, the aviation industry, may place further
restrictions on chemicals used for fuel treatment.
6. Apparatus
6.1 Colony Counter—Any of several types, for example, a Quebec Colony Counter may be used.
6.2 Drums; Steel—208 L (55 gal) 16 ga. steel, open-head drum with removable 16 ga. lid fitted with 2.05 cm and 1.90 cm 1.90 cm
threaded ports for venting and sampling.
6.3 Incubator—Any incubator capable of maintaining temperature of 3030 °C to 35 °C may be used.
6.4 Glass Jars—French square or similar configuration.
NOTE 1—Jar capacity should be determined based on the test plan designed fuel to water ratio and the expected sample volume size needed for weekly
testing (9.5 and 9.9).
6.5 Pails; Steel—18.9 L (5 gal) steel, open-head pail with removable 16 ga. lid fitted with 2.05 cm and 1.90 cm threaded ports
for venting and sampling.
6.6 Sterilizer—Any suitable steam sterilizer capable of producing the conditions of sterility is acceptable. A pressurized filter
sterilization apparatus of appropriate capacity to filter sterilize the test fuels and bottom-water used in the negative control
microcosms. A 0.2 μm pore-size methyl cellulose or cellulose acetate membrane should be used as the filtration medium.
6.7 Vortex—Mixer.
E1259 − 23
7. Reagents and Materials
7.1 Petri Dishes—100 by 15 mm 100 mm by 15 mm required for performing standard plate count.
7.2 Bacteriological Pipets—10.0 mL and 1.1, or 2.2 mL 1.1 mL, or 2.2 mL capacity.
7.3 Water Dilution Bottles—Any sterilizable glass container having a 150 to 200 mL 150 mL to 200 mL capacity and tight closure
may be used.
7.4 Fuel.
NOTE 2—Representative fuel samples from each product grade are available from all petroleum refiners.
7.5 Synthetic Bottom Water.
NOTE 3—In order to promote microbial growth of the inoculum when using the fuel as the sole source of organic nutrients, synthetic bottom water may
contain various inorganic nutrients. An example, of a commonly used synthetic bottom water is Bushnell-Haas Mineral Salts medium (BHMSS). with
the concentration adjusted to simulate a particular type of bottoms-water (marine, brackish, fresh, etc.).
7.6 Soy Peptone Casein Digest Agar.
7.7 Sabouraud Dextrose Agar.
7.8 Agar, Bacteriological Grade.
7.9 Potassium Tellurite Solution—sterile 1 %.
7.10 Gentamicin Sulfate—50 μg/mL.
7.11 Plate Count Agar.
7.12 Potato Dextrose Agar.
NOTE 4—Items 7.5 – 7.12 are available from a variety of media manufacturers and chemical supply companies.
8. Inoculum
8.1 Inoculum Selection:
8.1.1 Depending on the objectives of a test plan, one or more characterized cultures (for example: bacterium, yeast and mold) can
be selected or microbially contaminated bottoms-water collected from a fuel system can be used.
8.1.2 Contaminated fuel system microbial communities can be quite diverse and contain >50 different taxa. Consequently, when
Practice E1259 is to be used in order to assess a product’s general antimicrobial performance properties in fuel systems, multi-taxa
inocula provide a more realistic challenge population than either single or commonly used, three taxa inocula.
8.1.3 The use of standardized cultures to prepare microcosm inocula facilitates corroborative testing.
8.1.4 Inoculum taxa should be selected from cultures known to grow using fuel as their sole carbon source.
8.1.5 Depending on microcosm design, it can be appropriate to include aerobic and anaerobic taxa. If inhibition of
ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference Materials, American Chemical Society, Washington, DC. For
suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and
the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
Bushnell, L.D. and Haas, H.F. 1941. The utilization of certain hydrocarbons by microorganisms. J. Bacteriol. 41: 653- 673.
E1259 − 23
microbiologically influence corrosion is to be assessed, the challenge population should include iron related bacteria, acid
producing bacteria and sulfate reducing bacteria as part of the inoculum mixture.
8.1.6 Uncharacterized, bottoms-water, contaminant populations are most appropriate when Practice E1259 is to be used to
evaluate microbicide performance efficacy in a single system or family of systems (for example, bulk storage tanks for a specific
fuel grade at a specific facility).
8.2 Inoculum Preparation and Maintenance:
8.2.1 Inoculum Revitalization—Commonly used cultures are Pseudomonas aeruginosa, ATCC No. 33988, Hormoconis resinae,
ATCC No. 20495, and Candida viswanathii (formerly Candida tropicalis and Yarrowia tropicalis), ATCC No. 48138. However,
in accordance with 8.1, additional cultures can be used.
8.2.1.1 Obtain cultures from ATCC. Before initiating fuel antimicrobial tests, revitalize each of the three cultures in accordance
with the instructions contained with each culture.
8.2.2 Maintenance and Preparation of Pre-Inocula—All cultures are transferred from slants of a specified agar, (for example, a)
Pseudomonas aeruginosa (Plate Count Agar), (b) Hormoconis resinae Potato Dextrose Agar), and (c) Yarrowia tropicali (Potato
Dextrose Agar)) to synthetic bottom water medium in a suitable size screw-cap glass bottle (6.4).
8.2.2.1 Overlay inoculated bottom water with fuel to give a final fuel to water ratio of 10.
8.2.2.2 Keep this two-phase system at room temperature (20(20 °C to 30 °C) for seven days.
8.2.2.3 Weekly, transfer the interface, along with half the bottom water to a similar system until the inoculum used.
8.2.2.4 During this inoculum preparation period the bacterial levels should be maintained at approximately 10 CFU/mL or
6 4
non-culture test bioburden equivalent, the yeast levels at approximately 10 CFU/mL, and mold levels at approximately 10
spores/mL.
8.2.2.5 Freshly collected, microbially contaminated bottoms-water can be maintained per 8.2.2.1 – 8.2.2.4.
8.2.3 Preparation of Challenge (Test) Inoculum:
8.2.3.1 To prepare the test inoculum, dilute bacterial pre-inocula 1:100 to achieve a population equivalent to approximately 10
CFU/mL. Dilute yeast and molds 1:10 to achieve a population equivalent to approximately 10 CFU/mL.
8.2.3.2 At time zero, just prior to adding inoculum to each setup, and at each subsequent time point, determine the microbial
population density (9.9).
8.2.3.3 If test systems larger than 1.0 L will be used, the challenge inoculum should first be acclimated to growth in systems that
contain the same volume and fuel to bottom-water ratio as the test systems.
9. Procedure
9.1 Test Array Determination—The test plan determines the number and capacities of microcosms needed for the test plan.
Preferably, dupli
...