Standard Test Method for Screening of Fuels and Fuel Associated Aqueous Specimens for Microbial Contamination by Lateral Flow Immunoassay
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 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).
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 aerobic microorganisms during active growth in fuels.
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.
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.
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