ASTM D8070-23

Standard Test Method for Screening of Fuels and Fuel Associated Aqueous Specimens for Microbial Contamination by Lateral Flow Immunoassay

SIGNIFICANCE AND USE
5.1 This test method is intended to provide a tool for assessing whether fuel storage and distribution facilities, or end user fuel tanks, are subject to microbial growth, and to alert fuel suppliers or users to the potential for fuel quality or operational problems or the requirement for preventative or remedial measures, or both.  
5.2 This test method allows assessment of whether antigens generated by microbial activity in the specimens are present within specific defined ranges.  
5.3 This test method measures the presence of microbial and metabolite antigens in a specimen. The antigens are generated from the living cells and metabolites created by fungi and bacteria during growth on fuel. Consequently, the presence of antigens is an indicator of microbial contamination in fuel systems. Antigens are not associated with matter of nonbiological origin.  
5.3.1 Some of the antigens detected by this test method can persist after treatment with a biocide. See 11.4.  
5.4 This test method is semi-quantitative and can be used to determine whether contamination in samples drawn from fuel tanks and systems is negligible or present at moderate or heavy levels.  
5.4.1 Further information on using the test to assess biodeterioration risk is provided in Appendix X1.  
5.5 The significance of these levels to the operator will depend on the fuel type, the sampling location, the equipment or facility sampled, and the specific operating circumstances.  
5.6 Further guidance on interpretation of test results can be found in Guide D6469, in Energy Institute guidelines for the investigation of the microbial content of petroleum fuels, and in the IATA Guidance Material on Microbial Contamination in Aircraft Fuel Tanks.  
5.7 Further guidance on sampling can be found in Practice D7464.  
5.8 Testing can be conducted on a routine basis or to investigate incidents.  
5.9 Microbiological tests are not intended to be used to determine compliance with fuel specifications or lim...
SCOPE
1.1 This test method describes a procedure that can be used in the field or in a laboratory to detect antigens indicative of microbial contamination in liquid fuels, including those blended with synthesized hydrocarbons or biofuels, with kinematic viscosities (at 40 °C) of ≤24 mm2s–1 (for example, Specifications D396, D975, and D1655) and in fuel-associated water.  
1.1.1 This test method has been validated by an ILS for a range of middle distillate fuels meeting Specification D1655, EN590, Specification D975, and ISO 8217:2012.  
1.2 This test method semi-quantitatively assesses the concentration of specific antigens generated by commonly recovered, fuel-associated, aerobic microorganisms during active growth in fuel systems.  
1.2.1 A proprietary formulation of antibodies and antibody mixtures is used to detect three types of microbial antigen contamination: antigens generally found in aerobic bacteria, antigens generally present in common fungi (yeast and molds), and an antigen that is characteristic of Hormoconis resinae (the fungus most commonly associated with fuel biodeterioration).  
1.2.2 Although the antibodies and antibody mixtures are characteristic of diverse types of bacteria and fungi, it is unlikely that they are universal. Recognizing that for every microbe that has been isolated and characterized, it is likely that there are a billion that have not. Consequently, as is the case with all microbiological test methods, this test method does not purport to detect 100 % of the microbes present in a fuel or fuel-associated water sample.  
1.3 For each of the three sets of antigen detected (H. resinae, common fungi, and aerobic bacteria), the test detects whether the antigen concentration present is within set ranges representing negligible, moderate, or heavy microbial contamination.  
1.3.1 For fuel specimens, the antigen concentration ranges detected are 750 µg/L (heavy).  
1.3.2 For specimens of wat...

General Information

• Status: Published
• Publication Date: 30-Jun-2023
• ICS: 75.160.20 - Liquid fuels
• Technical Committee: D02 - Petroleum Products, Liquid Fuels, and Lubricants
• Drafting Committee: D02.14 - Stability, Cleanliness and Compatibility of Liquid Fuels

Relations

• Refers: ASTM D1655-24 - Standard Specification for Aviation Turbine Fuels
• Effective Date: 15-Mar-2024
• Refers: ASTM D975-23a - Standard Specification for Diesel Fuel
• Effective Date: 15-Dec-2023
• Refers: ASTM D4175-23a - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 15-Dec-2023
• Refers: ASTM D1655-23a - Standard Specification for Aviation Turbine Fuels
• Effective Date: 01-Oct-2023
• Refers: ASTM D4175-23e1 - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Jul-2023
• Refers: ASTM D6974-20 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-May-2020
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D7464-20 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-May-2020
• Refers: ASTM D396-19a - Standard Specification for Fuel Oils
• Effective Date: 15-Dec-2019
• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM D7464-19 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-Nov-2019
• Refers: ASTM D396-19 - Standard Specification for Fuel Oils
• Effective Date: 01-May-2019
• Refers: ASTM D975-19 - Standard Specification for Diesel Fuel Oils
• Effective Date: 01-Feb-2019
• Refers: ASTM D975-18a - Standard Specification for Diesel Fuel Oils
• Effective Date: 01-Dec-2018
• Refers: ASTM D396-18a - Standard Specification for Fuel Oils
• Effective Date: 01-Oct-2018

Overview

ASTM D8070-23 is the Standard Test Method for Screening of Fuels and Fuel Associated Aqueous Specimens for Microbial Contamination by Lateral Flow Immunoassay, published by ASTM International. This method provides a semi-quantitative procedure for detecting microbial antigens that indicate microbial contamination in liquid fuels-including diesel, aviation turbine fuel, marine fuels, and fuel-associated water-using lateral flow immunoassay (LFD) technology. The method addresses both bacteria and fungi (notably Hormoconis resinae, commonly associated with fuel biodeterioration), making it a critical part of routine fuel quality monitoring and maintenance.

Microbial contamination in fuels and fuel systems represents a significant risk to storage integrity, operational reliability, and fuel quality. By utilizing ASTM D8070-23, operators can screen fuel systems in the field or laboratory for signs of biodeterioration, thus enabling timely preventative or remedial action.

Key Topics

• Detection Method: Utilizes a series of lateral flow devices (LFDs) containing proprietary antibodies to detect antigens derived from bacteria, common fungi (yeasts and molds), and H. resinae.
• Specimen Types: Applicable to liquid fuels (including biofuel blends and synthesized hydrocarbons, with viscosities ≤24 mm²/s at 40°C) and fuel-associated aqueous phases.
• Operational Simplicity: The test can be conducted both in the field and laboratory settings. It does not require specialist microbiological training, nor an aseptic environment.
• Contamination Assessment Ranges:
  • For fuels: Negligible (<150 µg/L), Moderate (150 to 750 µg/L), and Heavy (>750 µg/L) microbial antigen concentrations.
  • For water: Negligible (<33 µg/mL), Moderate (33 to 166 µg/mL), and Heavy (>166 µg/mL) microbial antigen concentrations.
• Rapid Results: Delivers results within 15 minutes, allowing for prompt decision-making regarding fuel system management.
• Reliability: Designed for consistent, repeatable screening of microbial contamination, suited for both routine surveillance and incident investigations.

Applications

ASTM D8070-23 is widely applicable across industries where fuel quality and equipment reliability are critical, including:

• Fuel Storage and Distribution: Monitoring and maintenance of bulk storage tanks, pipelines, distribution vehicles, and end-user tanks for microbial contamination.
• Aviation: Screening aviation turbine fuel (Jet A, Jet A1, etc.) and onboard fuel tanks for microbial growth, aligning with requirements from authorities such as IATA.
• Marine: Assessing marine diesel and fuel-associated water in compliance with global shipping standards (e.g., ISO 8217) to prevent microbial fouling and engine issues.
• Industrial Power and Backup Generators: Routine surveillance of diesel generator tanks to preempt operational interruptions.
• Biofuels and Blends: Evaluating the microbial stability of biodiesel and fuel blends, which are especially susceptible to fungal and bacterial growth.
• Regulatory Compliance, Maintenance, and Risk Assessment: Integrates with facility inspection routines and forms part of a comprehensive fuel quality monitoring program.

Related Standards

Operators and laboratories using ASTM D8070-23 should also be familiar with the following related standards for comprehensive fuel system management:

• ASTM D6469: Guide for Microbial Contamination in Fuels and Fuel Systems
• ASTM D7464: Practice for Manual Sampling of Liquid Fuels for Microbiological Testing
• ASTM D396, D975, D1655: Specifications for Fuel Oils, Diesel, and Aviation Turbine Fuels
• ISO 8217: Specifications of Marine Fuels
• EN 590: Standard for Diesel Fuel
• IATA Guidance Material: Microbial Contamination in Aircraft Fuel Tanks
• ASTM D7687, D7978: Alternative test methods for microbial content
• ASTM D4176: Test Method for Free Water and Particulate Contamination

Practical Value

• Prevention of Fuel System Failures: Early detection of microbial contamination reduces the risk of filter blockages, corrosion, fuel degradation, and system outages.
• Optimized Maintenance: Enables proactive, condition-based fuel system cleaning and treatment, rather than relying on reactive measures.
• Efficient and User-Friendly: Fast, straightforward workflow with semi-quantitative results supports prompt and informed maintenance decisions in diverse operational environments.
• Supports Compliance and Safety: Aligns with industry guidelines and regulatory requirements for both routine and investigative testing.

By implementing ASTM D8070-23, organizations can ensure fuel quality, protect assets, reduce operational downtime, and comply with industry best practices for microbial contamination control.

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: D8070 − 23
Standard Test Method for
Screening of Fuels and Fuel Associated Aqueous
Specimens for Microbial Contamination by Lateral Flow
Immunoassay
This standard is issued under the fixed designation D8070; 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* 1.3.1 For fuel specimens, the antigen concentration ranges
detected are <150 μg ⁄L (negligible), 150 μg ⁄L to 750 μg ⁄L
1.1 This test method describes a procedure that can be used
(moderate), and >750 μg ⁄L (heavy).
in the field or in a laboratory to detect antigens indicative of
1.3.2 For specimens of water associated with fuel, the
microbial contamination in liquid fuels, including those
antigen concentration ranges detected are <33 μg ⁄mL
blended with synthesized hydrocarbons or biofuels, with kine-
2 –1 (negligible), 33 μg ⁄mL to 166 μg ⁄mL (moderate), and
matic viscosities (at 40 °C) of ≤24 mm s (for example,
>166 μg ⁄mL (heavy).
Specifications D396, D975, and D1655) and in fuel-associated
water. 1.4 The values stated in SI units are to be regarded as
1.1.1 This test method has been validated by an ILS for a standard. No other units of measurement are included in this
range of middle distillate fuels meeting Specification D1655, standard.
EN590, Specification D975, and ISO 8217:2012.
1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.2 This test method semi-quantitatively assesses the con-
responsibility of the user of this standard to establish appro-
centration of specific antigens generated by commonly
priate safety, health, and environmental practices and deter-
recovered, fuel-associated, aerobic microorganisms during ac-
mine the applicability of regulatory limitations prior to use.
tive growth in fuel systems.
For a specific hazard statement, see Section 8.
1.2.1 A proprietary formulation of antibodies and antibody
1.6 This international standard was developed in accor-
mixtures is used to detect three types of microbial antigen
dance with internationally recognized principles on standard-
contamination: antigens generally found in aerobic bacteria,
ization established in the Decision on Principles for the
antigens generally present in common fungi (yeast and molds),
Development of International Standards, Guides and Recom-
and an antigen that is characteristic of Hormoconis resinae (the
mendations issued by the World Trade Organization Technical
fungus most commonly associated with fuel biodeterioration).
Barriers to Trade (TBT) Committee.
1.2.2 Although the antibodies and antibody mixtures are
characteristic of diverse types of bacteria and fungi, it is
2. Referenced Documents
unlikely that they are universal. Recognizing that for every
microbe that has been isolated and characterized, it is likely
2.1 ASTM Standards:
that there are a billion that have not. Consequently, as is the
D396 Specification for Fuel Oils
case with all microbiological test methods, this test method
D975 Specification for Diesel Fuel
does not purport to detect 100 % of the microbes present in a
D1129 Terminology Relating to Water
fuel or fuel-associated water sample.
D1655 Specification for Aviation Turbine Fuels
D4175 Terminology Relating to Petroleum Products, Liquid
1.3 For each of the three sets of antigen detected (H.
Fuels, and Lubricants
resinae, common fungi, and aerobic bacteria), the test detects
D4176 Test Method for Free Water and Particulate Contami-
whether the antigen concentration present is within set ranges
nation in Distillate Fuels (Visual Inspection Procedures)
representing negligible, moderate, or heavy microbial contami-
D6469 Guide for Microbial Contamination in Fuels and Fuel
nation.
Systems
This test method is under the jurisdiction of ASTM Committee D02 on
Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of
Subcommittee D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved July 1, 2023. Published July 2023. Originally published contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
in 2016. Last previous edition approved in 2021 as D8070 – 21. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
D8070-23. the ASTM website.
*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
D8070 − 23
D6974 Practice for Enumeration of Viable Bacteria and 3.2.5 colony, n—a discreet visible aggregate of microorgan-
Fungi in Liquid Fuels—Filtration and Culture Procedures isms that develops when a viable microorganism, or particle
D7464 Practice for Manual Sampling of Liquid Fuels, As- containing viable microorganisms, is introduced into a gel-
sociated Materials and Fuel System Components for based nutritive culture medium and reproduces there.
Microbiological Testing
3.2.6 colony forming unit (CFU), n—a viable microorgan-
D7687 Test Method for Measurement of Cellular Adenosine
ism or aggregate of viable microorganisms, which prolifer-
Triphosphate in Fuel and Fuel-associated Water With
ate(s) in a culture medium to produce a viable colony.
Sample Concentration by Filtration
3.2.7 lateral flow device, n—in immunology, an antibody-
D7847 Guide for Interlaboratory Studies for Microbiological
impregnated, porous medium through which an antigen-
Test Methods
containing buffer is permitted to wick in order to bring the
D7978 Test Method for Determination of the Viable Aerobic
antigen into contact with the antibody.
Microbial Content of Fuels and Associated Water—
Thixotropic Gel Culture Method
3.2.7.1 Discussion—Typically, the antibody is linked to an
E1326 Guide for Evaluating Non-culture Microbiological
indicator which produces a color reaction when antibody and
Tests
antigen combine.
E2756 Terminology Relating to Antimicrobial and Antiviral
3.2.8 metabolite, n—a chemical substance produced by any
Agents
of the many complex chemical and physical processes involved
2.2 Other Standards:
in the maintenance of life.
BS EN590 Standard for Diesel Fuel
3.2.9 microorganisms, n—organisms too small to be seen
ISO 8217:2012 Petroleum products—Fuels (Class F)—
with the naked eye, which generally include bacteria, protozoa,
Specifications of marine fuels
fungi, and microalgae (sometimes collectively called slime or
NATO Logistics Handbook Chapter 15: Fuels, Oils, Lubri-
microbial contamination).
cants and Petroleum Handling Equipment, Annex A: Aide
Memoire on Fuels in NATO
3.2.9.1 Discussion—In the context of this test method,
IATA Guidance Material on Microbiological Contamination
microorganisms are bacteria and fungi (yeasts and molds) that
in Aircraft Fuel Tanks, Fifth Edition, 2015
are capable of growth in fuels and associated aqueous-phase
fluid.
3. Terminology
3.3 Definitions of Terms Specific to This Standard:
3.1 Definitions:
3.3.1 extraction fluid, n—a mixture of buffer and surfactants
3.1.1 For definitions of terms used in this test method, refer
used to extract antigens from the specimens.
to Terminologies D1129, D4175, E2756, and Guide E1326.
3.3.2 surfactant (surface active agent), n—a substance that
3.2 Definitions:
affects the interfacial or surface tension of solutions even when
3.2.1 aerobic, adj—(1) taking place in the presence of
present in very low concentrations.
oxygen, (2) living or active in the presence of oxygen.
3.4 Acronyms:
3.2.2 antibody, n—an immunoglobulin, a protein that is
produced as a part of the immune response which is capable of 3.4.1 CFU—colony forming unit
specifically combining with the antigen.
3.4.2 FSII—fuel system icing inhibitor
3.2.2.1 Discussion—In the context of this test method,
3.4.3 LFD—lateral flow device
antibodies created for this purpose are utilized in conjunction
with visual indicators to detect presence of microbial antigens.
4. Summary of Test Method
3.2.3 antigen, n—a substance that stimulates the host to
4.1 Microbial contamination is detected using a series of
produce an immune response. In the context of this test
antibodies immobilized onto a test tray in the form of three
method, specific antigens are detected as indicators of micro-
pairs of lateral flow devices (LFDs). These antibodies are used
bial contamination.
to detect antigens from common bacteria and fungi that
3.2.4 buffer, n—a compound or mixture that, when con-
proliferate in fuel tanks and systems.
tained in solution, causes the solution to resist change in pH.
4.1.1 The LFDs contain broad spectrum antibodies raised
3.2.4.1 Discussion—Each buffer has a characteristic limited
against cell components and materials generated during micro-
range of pH over which it is effective. bial growth on hydrocarbons (the antigens). These antibodies
indicate the presence of H. resinae, other common fungi, and
aerobic bacteria within concentration ranges described in 1.3.
Available from British Standards Institution (BSI), 389 Chiswick High Rd.,
London W4 4AL, U.K., http://www.bsigroup.com. 4.2 Fuel or aqueous specimens from fuel are mixed with an
Available from International Organization for Standardization (ISO), ISO
aqueous extraction fluid in an extraction bottle. The extraction
Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,
fluid captures antigenic material present in the specimen.
Geneva, Switzerland, http://www.iso.org.
Available from North Atlantic Treaty Organization (NATO) at http://
4.3 Four drops of the separated extraction fluid are dis-
www.nato.int/docu/logi-en/1997/lo-15a.htm.
pensed into the sample well of each of the six LFDs, which are
Available from International Aviation Transport Association (IATA) at http://
store.iata.org. arranged in three pairs.
D8070 − 23
4.4 For each of the three types of contamination (H. resinae, 5.6 Further guidance on interpretation of test results can be
other common fungi, and aerobic bacteria), a pair of LFDs found in Guide D6469, in Energy Institute guidelines for the
indicates the concentration range of the detected antigen investigation of the microbial content of petroleum fuels, and
present in the specimen. For each LFD pair, the minimum in the IATA Guidance Material on Microbial Contamination in
detection level is set so that one is at the lower boundary of the Aircraft Fuel Tanks.
moderate contamination range and the other at the lower
5.7 Further guidance on sampling can be found in Practice
boundary of the heavy contamination range of antigen concen-
D7464.
tration stated in 1.3.
5.8 Testing can be conducted on a routine basis or to
4.5 Presence of antigen below the minimum level of detec-
investigate incidents.
tion for each LFD is indicated by development of red control
and test lines. If only the control line becomes visible, then 5.9 Microbiological tests are not intended to be used to
antigen concentration in the specimen is above the minimum
determine compliance with fuel specifications or limits. The
level of detection for the LFD. When antigen is present at or implementation of specification limits for microbiological
above threshold concentrations, test lines on the LFD fail to contamination in fuels is generally not appropriate, and micro-
appear. By reading both LFDs in the pair, it can be established bial contamination levels cannot be used alone or directly to
which range the antigen concentration falls within, that is, make inferences about fuel quality or fitness for use.
negligible, moderate, or heavy.
5.10 When interpreting results, it must be appreciated that
4.6 The control lines appear within 10 min and the result is
the test result applies only to the specific sample and specimen
read at 15 min, although the lines are stable up to 30 min.
tested and not necessarily to bulk fuel. Microbiological con-
tamination usually shows a highly heterogeneous distribution
4.7 If contaminating antigens are not present, test lines will
in fuel systems, and therefore, analysis of a single sample will
also appear within 10 min and the result is read within 30 min
rarely provide a complete assessment of the overall levels of
(see 4.6). If test lines do not appear, then this indicates the
contamination present.
presence of contamination at the concentration ranges de-
scribed in 1.3.
5.11 Water phase will usually contain substantially higher
amounts of microbial contamination than fuel phase and,
4.8 The results and antigen concentration ranges are deter-
consequently, a different interpretation of results is required.
mined and recorded.
This is why this test method reports antigen concentration per
mL for water and per L for fuel.
5. Significance and Use
5.12 This test method differs from some other methods (for
5.1 This test method is intended to provide a tool for
example Test Methods D7687 and D7978) and practices (for
assessing whether fuel storage and distribution facilities, or end
example Practice D6974) in that it detects microbial activity in
user fuel tanks, are subject to microbial growth, and to alert
fuels or associated aqueous specimens in the field and does not
fuel suppliers or users to the potential for fuel quality or
need to be performed in a laboratory or in an aseptic environ-
operational problems or the requirement for preventative or
ment. It may be used in a laboratory.
remedial measures, or both.
5.13 This test method does not require specialist microbio-
5.2 This test method allows assessment of whether antigens
logical experience or knowledge.
generated by microbial activity in the specimens are present
within specific defined ranges.
5.14 This test method provides rapid results that reflect the
5.3 This test method measures the presence of microbial and total active microbial contamination in the specimen, and
enables result to be obtained within 15 min.
metabolite antigens in a specimen. The antigens are generated
from the living cells and metabolites created by fungi and
5.15 This test method differentiates among three ranges of
bacteria during growth on fuel. Consequently, the presence of
contamination for H. resinae, other fungi, and aerobic bacteria
antigens is an indicator of microbial contamination in fuel
(see 1.3).
systems. Antigens are not associated with matter of nonbio-
logical origin.
6. Interferences and Possible Test Method Errors
5.3.1 Some of the antigens detected by this test method can
6.1 Drops of the extraction fluid can fail to be expelled from
persist after treatment with a biocide. See 11.4.
the extraction bottle if particulate material in the sample blocks
5.4 This test method is semi-quantitative and can be used to
the dropper nozzle. The nozzle should be removed and cleared.
determine whether contamination in samples drawn from fuel
tanks and systems is negligible or present at moderate or heavy 6.2 If fuel inadvertently comes into contact with the LFD, it
levels. can prevent the control line developing and the test will be
5.4.1 Further information on using the test to assess biode- invalid. Contact of fuel specimen with the LFD must be
avoided.
terioration risk is provided in Appendix X1.
5.5 The significance of these levels to the operator will 6.3 Do not touch the LFD viewing windows. Touching the
depend on the fuel type, the sampling location, the equipment viewing windows can contaminate the LFD. If this occurs, the
or facility sampled, and the specific operating circumstances. test is invalid. Repeat steps 10.1 – 10.9.
D8070 − 23
6.4 Test line color intensity is affected by contamination 7.4 Disposable Pipet, Pasteur, 5 mL.
levels. Above the threshold levels of antigen concentration
NOTE 2—All of the above are contained within the FUELSTAT PLUS
(that is, above moderate and above heavy), the test line will not
test kits.
appear. However, as these concentrations are approached, the
line may be faint. A moderate or heavy level of contamination
8. Hazards
should not be reported unless the test line is not visible.
8.1 Hazards are typical of those experienced when handling
6.5 Test lines are visible when target antigens are present at
fuel. There are no additional hazards associated with this test
concentrations below the lower thresholds. When antigen
method.
concentrations are below detection limits (see 11.3.1), all lines
are visible and contamination levels are considered to be
9. Sampling
negligible.
9.1 Samples shall be collected and handled in accordance
6.6 Test lines on the high LFDs are visible at concentrations
with Practice D7464.
greater than the lower thresholds (>150 μg ⁄L for fuel samples
or >33 μg ⁄mL for water sampels) but less than the higher 9.2 A minimum sample of 150 mL of fuel, or fuel/aqueous
mix, or 15 mL of fuel-associated aqueous solution alone is
thresholds (≤750 μg ⁄L for fuel samples or ≤166 μg ⁄mL for
water samples) (1.3, X1.1). required to perform this test method.
6.7 If red control or test lines are visible before the
NOTE 3—As part of the overall analysis of the fuel, it is recommended
extraction fluid is added, the LFD might be damp or wet. that a visual inspection is undertaken before this test method is carried out,
using Test Method D4176.
Repeat the method with new LFD trays.
6.8 Interference by fuel system icing inhibitor (FSII), typi-
10. Procedure
cally diethyleneglycol monoethyl ether, is not normally an
10.1 Collect sample in accordance with 9.1.
issue, providing the concentration in the fuel or water phase
does not exceed levels typically encountered in treated fuels.
10.2 Shake sample for approximately 30 s and allow to
6.8.1 Excessive FSII concentrations can delay wicking from
settle for 12 min 6 1 min. Use separated water, fuel, or a
the normal 10 min (see 10.1) interval to as long as 1 h.
mixture of both, as described in 10.6.
6.8.2 When FSII is added to fuel at concentrations greater
NOTE 4—The required settling time will depend on from where in the
than those stipulated in most fuel specifications, or when very
fuel phase the specimen for test is withdrawn after shaking. For example,
high concentrations of FSII partition to a sample water phase
a typical 200 mL sample allowed to settle for 12 min will theoretically
tested, or both occur, very high concentrations of FSII can be
have no suspended free water remaining in any fuel phase. However, a 1 L
captured in the extraction fluid.
sample settled for the same time will still have suspended water in most
of the fuel. Test results are likely to vary based on the location from within
6.8.3 This test method is still usable when FSII interference
the partially phase-separated sample and the means by which the test
is present, but if LFD control lines are not visible (see 10.11),
specimen is retrieved.
LFD should be observed at 10 min intervals for up to 1 h.
10.3 The polyethylene sample extraction bottle has a flat
6.8.4 If LFD control lines (see 10.11) are not visible within
transit cap, unscrew and remove this (see Fig. 1).
1 h, the test results are not valid
6.9 Additives designed to retain water in suspension or 10.4 Remove LFD tray from foil pouch and label the LFD
diesel fuels containing high levels of FAME might delay rate of tray (Fig. 2) with specimen details and date of collection.
extraction fluid separation but will have no effect on the run
NOTE 5—LFD rows on the tray are labeled ‘high’ and ‘low.’ This is a
time of the test or the results. See Note 4 in 10.2.
prompt to remind which row should be read to indicate lower contami-
nation levels and which for higher.
7. Reagents and Materials
10.5 Visually inspect the sample to identify if water is
7.1 Antigen Extraction Fluid
present in accordance with the procedures described in Test
7.2 LFD Tray—integrated assembly containing a tray of six
Method D4176 (see 10.2).
paired LFDs for the detection of H. resinae, other fungi, and
10.6 Extraction—Select from extraction procedure A, B, or
bacteria.
C, based on volume of visible water in sample:
NOTE 1—Each pair of LFDs includes one LFD to detect intermediate
10.6.1 Extraction Procedure A—If there is ≥15 mL water in
antigen concentrations (1.3) and one to detect antigen concentrations at
the sample, transfer this water into the polyethylene sample
the upper detection limit (1.3).
extraction bottle (10.3) to the water phase line.
7.3 Polyethylene Extraction Bottle, HDPE, 175 mL with a
10.6.2 Extraction Procedure B—If there is visible water
dropper nozzle delivering 0.1 mL per drop.
present, but is <15 mL, transfer all of the water into the
polyethylene sample extraction bottle (10.3) and then transfer
fuel from the sample up to the fuel phase line.
The sole source of supply of the LFD Extraction Fluid and FUELSTAT PLUS
(trademarked) test kits known to the committee at this time is Conidia Bioscience
NOTE 6—Appendix X5 provides instructions for the use of a disposable
Ltd., Egham, TW20 9TY, UK. If you are aware of alternative suppliers please
syringe and an extraction tube to
...

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: D8070 − 21 D8070 − 23
Standard Test Method for
Screening of Fuels and Fuel Associated Aqueous
Specimens for Microbial Contamination by Lateral Flow
Immunoassay
This standard is issued under the fixed designation D8070; 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 test method describes a procedure that can be used in the field or in a laboratory to detect antigens indicative of microbial
contamination in liquid fuels, including those blended with synthesized hydrocarbons or biofuels, with kinematic viscosities (at
2 –1
40 °C) of ≤24 mm s (for example, Specifications D396, D975, and D1655) and in fuel-associated water.
1.1.1 This test method has been validated by an ILS for a range of middle distillate fuels meeting Specification D1655, EN590,
Specification D975, and ISO 8217:2012.
1.2 This test method semi-quantitatively assesses the concentration of specific antigens generated by commonly recovered,
fuel-associated, aerobic microorganisms during active growth in fuels.fuel systems.
1.2.1 A proprietary formulation of antibodies and antibody mixtures is used to detect three types of microbial antigen
contamination: antigens generally found in aerobic bacteria, antigens generally present in common fungi (yeast and molds), and
an antigen that is characteristic of Hormoconis resinae (the fungus most commonly associated with fuel biodeterioration).
1.2.2 Although the antibodies and antibody mixtures are characteristic of diverse types of bacteria and fungi, it is unlikely that
they are universal. Recognizing that for every microbe that has been isolated and characterized, it is likely that there are a billion
that have not. Consequently, as is the case with all microbiological test methods, this test method does not purport to detect 100 %
of the microbes present in a fuel or fuel-associated water sample.
1.3 For each of the three sets of antigen detected (H. resinae, common fungi, and aerobic bacteria), the test detects whether the
antigen concentration present is within set ranges representing negligible, moderate, or heavy microbial contamination.
1.3.1 For fuel specimens, the antigen concentration ranges detected are <150 μg ⁄L (negligible), 150 μg ⁄L to 750 μg ⁄L (moderate),
and >750 μg ⁄L (heavy).
1.3.2 For specimens of water associated with fuel, the antigen concentration ranges detected are <33 μg ⁄mL (negligible),
33 μg ⁄mL to 166 μg ⁄mL (moderate), and >166 μg ⁄mL (heavy).
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
This test method is under the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcommittee
D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels.
Current edition approved April 1, 2021July 1, 2023. Published April 2021July 2023. Originally published in 2016. Last previous edition approved in 20202021 as
D8070 – 20.D8070 – 21. DOI: 10.1520/D8070-21.10.1520/D8070-23.
*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
D8070 − 23
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. For a specific hazard statement, see Section 8.
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
D975 Specification for Diesel Fuel
D1129 Terminology Relating to Water
D1655 Specification for Aviation Turbine Fuels
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D4176 Test Method for Free Water and Particulate Contamination in Distillate Fuels (Visual Inspection Procedures)
D6469 Guide for Microbial Contamination in Fuels and Fuel Systems
D6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
D7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological
Testing
D7687 Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel and Fuel-associated Water With Sample
Concentration by Filtration
D7847 Guide for Interlaboratory Studies for Microbiological Test Methods
D7978 Test Method for Determination of the Viable Aerobic Microbial Content of Fuels and Associated Water—Thixotropic Gel
Culture Method
E1326 Guide for Evaluating Non-culture Microbiological Tests
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 Other Standards:
BS EN590 Standard for Diesel Fuel
ISO 8217:2012 Petroleum products—Fuels (Class F)—Specifications of marine fuels
NATO Logistics Handbook Chapter 15: Fuels, Oils, Lubricants and Petroleum Handling Equipment, Annex A: Aide Memoire
on Fuels in NATO
IATA Guidance Material on Microbiological Contamination in Aircraft Fuel Tanks, Fifth Edition, 2015
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this test method, refer to Terminologies D1129, D4175, E2756, and Guide E1326.
3.2 Definitions:
3.2.1 aerobic, adj—(1) taking place in the presence of oxygen, (2) living or active in the presence of oxygen.
3.2.2 antibody, n—an immunoglobulin, a protein that is produced as a part of the immune response which is capable of specifically
combining with the antigen.
3.2.2.1 Discussion—
In the context of this test method, antibodies created for this purpose are utilized in conjunction with visual indicators to detect
presence of microbial antigens.
3.2.3 antigen, n—a substance that stimulates the host to produce an immune response. In the context of this test method, specific
antigens are detected as indicators of microbial contamination.
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.
Available from British Standards Institution (BSI), 389 Chiswick High Rd., London W4 4AL, U.K., http://www.bsigroup.com.
Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva,
Switzerland, http://www.iso.org.
Available from North Atlantic Treaty Organization (NATO) at http://www.nato.int/docu/logi-en/1997/lo-15a.htm.
Available from International Aviation Transport Association (IATA) at http://store.iata.org.
D8070 − 23
3.2.4 buffer, n—a compound or mixture that, when contained in solution, causes the solution to resist change in pH.
3.2.4.1 Discussion—
Each buffer has a characteristic limited range of pH over which it is effective.
3.2.5 colony, n—a discreet visible aggregate of microorganisms that develops when a viable microorganism, or particle containing
viable microorganisms, is introduced into a gel-based nutritive culture medium and reproduces there.
3.2.6 colony forming unit (CFU), n—a viable microorganism or aggregate of viable microorganisms, which proliferate(s) in a
culture medium to produce a viable colony.
3.2.7 lateral flow device, n—in immunology, an antibody-impregnated, porous medium through which an antigen-containing
buffer is permitted to wick in order to bring the antigen into contact with the antibody.
3.2.7.1 Discussion—
Typically, the antibody is linked to an indicator which produces a color reaction when antibody and antigen combine.
3.2.8 metabolite, n—a chemical substance produced by any of the many complex chemical and physical processes involved in the
maintenance of life.
3.2.9 microorganisms, n—organisms too small to be seen with the naked eye, which generally include bacteria, protozoa, fungi,
and microalgae (sometimes collectively called slime or microbial contamination).
3.2.9.1 Discussion—
In the context of this test method, microorganisms are bacteria and fungi (yeasts and molds) that are capable of growth in fuels
and associated aqueous-phase fluid.
3.3 Definitions of Terms Specific to This Standard:
3.3.1 extraction fluid, n—a mixture of buffer and surfactants used to extract antigens from the specimens.
3.3.2 surfactant (surface active agent), n—a substance that affects the interfacial or surface tension of solutions even when present
in very low concentrations.
3.4 Acronyms:
3.4.1 CFU—colony forming unit
3.4.2 FSII—fuel system icing inhibitor
3.4.3 LFD—lateral flow device
4. Summary of Test Method
4.1 Microbial contamination is detected using a series of antibodies immobilized onto a test tray in the form of three pairs of lateral
flow devices (LFDs). These antibodies are used to detect antigens from common bacteria and fungi that proliferate in fuel tanks
and systems.
4.1.1 The LFDs contain broad spectrum antibodies raised against cell components and materials generated during microbial
growth on hydrocarbons (the antigens). These antibodies indicate the presence of H. resinae, other common fungi, and aerobic
bacteria within concentration ranges described in 1.3.
4.2 Fuel or aqueous specimens from fuel are mixed with an aqueous extraction fluid in an extraction bottle. The extraction fluid
captures antigenic material present in the specimen.
4.3 Four drops of the separated extraction fluid are dispensed into the sample well of each of the six LFDs, which are arranged
in three pairs.
D8070 − 23
4.4 For each of the three types of contamination (H. resinae, other common fungi, and aerobic bacteria), a pair of LFDs indicates
the concentration range of the detected antigen present in the specimen. For each LFD pair, the minimum detection level is set so
that one is at the lower boundary of the moderate contamination range and the other at the lower boundary of the heavy
contamination range of antigen concentration stated in 1.3.
4.5 Presence of antigen below the minimum level of detection for each LFD is indicated by development of red control and test
lines. If only the control line becomes visible, then antigen concentration in the specimen is above the minimum level of detection
for the LFD. When antigen is present at or above threshold concentrations, test lines on the LFD fail to appear. By reading both
LFDs in the pair, it can be established which range the antigen concentration falls within, that is, negligible, moderate, or heavy.
4.6 The control lines appear within 10 min and the result is read at 15 min, although the lines are stable up to 30 min.
4.7 If contaminating antigens are not present, test lines will also appear within 10 min and the result is read within 30 min (see
4.6). If test lines do not appear, then this indicates the presence of contamination at the concentration ranges described in 1.3.
4.8 The results and antigen concentration ranges are determined and recorded.
5. Significance and Use
5.1 This test method is intended to provide a tool for assessing whether fuel storage and distribution facilities, or end user fuel
tanks, are subject to microbial growth, and to alert fuel suppliers or users to the potential for fuel quality or operational problems
or the requirement for preventative or remedial measures, or both.
5.2 This test method allows assessment of whether antigens generated by microbial activity in the specimens are present within
specific defined ranges.
5.3 This test method measures the presence of microbial and metabolite antigens in a specimen. The antigens are generated from
the living cells and metabolites created by fungi and bacteria during growth on fuel. Consequently, the presence of antigens is an
indicator of microbial contamination in fuel systems. Antigens are not associated with matter of nonbiological origin.
5.3.1 Some of the antigens detected by this test method can persist after treatment with a biocide. See 11.4.
5.4 This test method is semi-quantitative and can be used to determine whether contamination in samples drawn from fuel tanks
and systems is negligible or present at moderate or heavy levels.
5.4.1 Further information on using the test to assess biodeterioration risk is provided in Appendix X1.
5.5 The significance of these levels to the operator will depend on the fuel type, the sampling location, the equipment or facility
sampled, and the specific operating circumstances.
5.6 Further guidance on interpretation of test results can be found in Guide D6469, in Energy Institute guidelines for the
investigation of the microbial content of petroleum fuels, and in the IATA Guidance Material on Microbial Contamination in
Aircraft Fuel Tanks.
5.7 Further guidance on sampling can be found in Practice D7464.
5.8 Testing can be conducted on a routine basis or to investigate incidents.
5.9 Microbiological tests are not intended to be used to determine compliance with fuel specifications or limits. The
implementation of specification limits for microbiological contamination in fuels is generally not appropriate, and microbial
contamination levels cannot be used alone or directly to make inferences about fuel quality or fitness for use.
5.10 When interpreting results, it must be appreciated that the test result applies only to the specific sample and specimen tested
D8070 − 23
and not necessarily to bulk fuel. Microbiological contamination usually shows a highly heterogeneous distribution in fuel systems,
and therefore, analysis of a single sample will rarely provide a complete assessment of the overall levels of contamination present.
5.11 Water phase will usually contain substantially higher amounts of microbial contamination than fuel phase and, consequently,
a different interpretation of results is required. This is why this test method reports antigen concentration per mL for water and per
L for fuel.
5.12 This test method differs from some other methods (for example Test Methods D7687 and D7978) and practices (for example
Practice D6974) in that it detects microbial activity in fuels or associated aqueous specimens in the field and does not need to be
performed in a laboratory or in an aseptic environment. It may be used in a laboratory.
5.13 This test method does not require specialist microbiological experience or knowledge.
5.14 This test method provides rapid results that reflect the total active microbial contamination in the specimen, and enables result
to be obtained within 15 min.
5.15 This test method differentiates among three ranges of contamination for H. resinae, other fungi, and aerobic bacteria (see 1.3).
6. Interferences and Possible Test Method Errors
6.1 Drops of the extraction fluid can fail to be expelled from the extraction bottle if particulate material in the sample blocks the
dropper nozzle. The nozzle should be removed and cleared.
6.2 If fuel inadvertently comes into contact with the LFD, it can prevent the control line developing and the test will be invalid.
Contact of fuel specimen with the LFD must be avoided.
6.3 Do not touch the LFD viewing windows. Touching the viewing windows can contaminate the LFD. If this occurs, the test is
invalid. Repeat steps 10.1 – 10.9.
6.4 Test line color intensity is affected by contamination levels. Above the threshold levels of antigen concentration (that is, above
moderate and above heavy), the test line will not appear. However, as these concentrations are approached, the line may be faint.
A moderate or heavy level of contamination should not be reported unless the test line is not visible.
6.5 Test lines are visible when target antigens are present at concentrations below the lower thresholds. When antigen
concentrations are below detection limits (see 11.3.1), all lines are visible and contamination levels are considered to be negligible.
6.6 The 750 μgTest lines⁄L and 166 μg on the⁄mL lines high LFDs are visible at concentrations greater than the lower thresholds
(>150 μg ⁄L for fuel samples or >33 μg ⁄mL for water sampels) but less than the higher thresholds.thresholds (≤750 μg ⁄L for fuel
samples or ≤166 μg ⁄mL for water samples) (1.3, X1.1).
6.7 If red control or test lines are visible before the extraction fluid is added, the LFD might be damp or wet. Repeat the method
with new LFD trays.
6.8 Interference by fuel system icing inhibitor (FSII), typically diethyleneglycol monoethyl ether, is not normally an issue,
providing the concentration in the fuel or water phase does not exceed levels typically encountered in treated fuels.
6.8.1 Excessive FSII concentrations can delay wicking from the normal 10 min (see 10.1) interval to as long as 1 h.
6.8.2 When FSII is added to fuel at concentrations greater than those stipulated in most fuel specifications, or when very high
concentrations of FSII partition to a sample water phase tested, or both occur, very high concentrations of FSII can be captured
in the extraction fluid.
6.8.3 This test method is still usable when FSII interference is present, but if LFD control lines are not visible (see 10.11), LFD
should be observed at 10 min intervals for up to 1 h.
D8070 − 23
6.8.4 If LFD control lines (see 10.11) are not visible within 1 h, the test results are not valid
6.9 Additives designed to retain water in suspension or diesel fuels containing high levels of FAME might delay rate of extraction
fluid separation but will have no effect on the run time of the test or the results. See Note 4 in 10.2.
7. Reagents and Materials
7.1 Antigen Extraction Fluid
7.2 LFD Tray—integrated assembly containing a tray of six paired LFDs for the detection of H. resinae, other fungi, and bacteria.
NOTE 1—Each pair of LFDs includes one LFD to detect intermediate antigen concentrations (1.3) and one to detect antigen concentrations at the upper
detection limit (1.3).
7.3 Polyethylene Extraction Bottle, HDPE, 175 mL with a dropper nozzle delivering 0.1 mL per drop.
7.4 Disposable Pipet, Pasteur, 5 mL.
NOTE 2—All of the above are contained within the FUELSTAT PLUS test kits.
8. Hazards
8.1 Hazards are typical of those experienced when handling fuel. There are no additional hazards associated with this test method.
9. Sampling
9.1 Samples shall be collected and handled in accordance with Practice D7464.
9.2 A minimum sample of 150 mL of fuel, or fuel/aqueous mix, or 15 mL of fuel-associated aqueous solution alone is required
to perform this test method.
NOTE 3—As part of the overall analysis of the fuel, it is recommended that a visual inspection is undertaken before this test method is carried out, using
Test Method D4176.
10. Procedure
10.1 Collect sample in accordance with 9.1.
10.2 Shake sample for approximately 30 s and allow to settle for 12 min 6 1 min. Use separated water, fuel, or a mixture of both,
as described in 10.6.
NOTE 4—The required settling time will depend on from where in the fuel phase the specimen for test is withdrawn after shaking. For example, a typical
200 mL sample allowed to settle for 12 min will theoretically have no suspended free water remaining in any fuel phase. However, a 1 L sample settled
for the same time will still have suspended water in most of the fuel. Test results are likely to vary based on the location from within the partially
phase-separated sample and the means by which the test specimen is retrieved.
10.3 The polyethylene sample extraction bottle has a flat transit cap, unscrew and remove this (see Fig. 1).
10.4 Remove LFD tray from foil pouch and label the LFD tray (Fig. 2) with specimen details and date of collection.
The sole source of supply of the LFD Extraction Fluid and FUELSTAT PLUS (trademarked) test kits known to the committee at this time is Conidia Bioscience Ltd.,
Egham, TW20 9TY, UK. If you are aware of alternative suppliers please provide this information to ASTM International Headquarters. Your comments will receive careful
consideration at a meeting of the responsible technical committee, which you may attend.
D8070 − 23
FIG. 1 Polyethylene Sample Extraction Bottle
FIG. 2 LFD Tray
NOTE 5—LFD rows on the tray are labeled ‘high’ and ‘low.’ This is a prompt to remind which row should be read to indicate lower contamination levels
and which for higher.
10.5 Visually inspect the sample to identify if water is present in accordance with the procedures described in Test Method D4176
(see 10.2).
10.6 Extraction—Select from extraction procedure A, B, or C, based on volume of visible water in sample:
10.6.1 Extraction Procedure A—If there is ≥15 mL water in the sample, transfer this water into the polyethylene sample extraction
bottle (10.3) to the water phase lineline.
...