1.1 该测试方法描述了一种可在现场或实验室中用于检测指示液体燃料中微生物污染的抗原的程序，包括那些与合成碳氢化合物或生物燃料混合的抗原，其运动粘度（40 °C） ≤ 24 毫米 2. s –1 （例如，规格 D396 ， D975 和 D1655 )以及在与燃料相关的水中。 1.1.1 该试验方法已通过ILS对一系列符合规范的中间馏分燃料进行了验证 D1655 ，EN590，规范 D975 和ISO 8217:2012。 1.2 该测试方法半定量评估了在燃料系统中活性生长期间，由通常回收的燃料相关需氧微生物产生的特异性抗原的浓度。 1.2.1 抗体和抗体混合物的专有配方用于检测三种类型的微生物抗原污染: 通常存在于需氧细菌中的抗原，通常存在于常见真菌（酵母和霉菌）中的抗原以及具有 树脂荷尔蒙介 （最常见的与燃料生物降解有关的真菌）。 1.2.2 尽管抗体和抗体混合物是不同类型细菌和真菌的特征，但它们不太可能具有普遍性。认识到每一种被分离和表征的微生物，很可能都有十亿种没有被分离和鉴定。因此，与所有微生物测试方法一样，该测试方法并不旨在检测100 % 存在于燃料或燃料相关水样中的微生物。 1.3 对于检测到的三组抗原中的每一组( 树脂H.resinae 常见真菌和需氧细菌），该测试检测存在的抗原浓度是否在代表可忽略、中度或重度微生物污染的设定范围内。 1.3.1 对于燃料样本，检测到的抗原浓度范围<150 µg/L（可忽略不计），150 µg/L至750 µg/L（中等），且>750 µg/L（重）。 1.3.2 对于与燃料相关的水样本，检测到的抗原浓度范围<33 µg/mL（可忽略不计），33 µg/mL至166 µg/mL（中等），且>166 µg/mL（重）。 1.4 以国际单位制表示的数值应视为标准。本标准中不包括其他计量单位。 1.5 本标准并非旨在解决与其使用相关的所有安全问题（如有）。 本标准的使用者有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 有关具体的危险说明，请参阅第节 8. 。 1.6 本国际标准是根据世界贸易组织技术性贸易壁垒委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认的标准化原则制定的。 ===意义和用途====== 5.1 该测试方法旨在提供一种工具，用于评估燃料储存和分配设施或最终用户燃料箱是否会受到微生物生长的影响，并提醒燃料供应商或用户潜在的燃料质量或操作问题，或预防或补救措施的要求，或两者兼而有之。 5.2 这种测试方法可以评估样本中微生物活性产生的抗原是否存在于特定的定义范围内。 5.3 该测试方法测量样本中微生物和代谢产物抗原的存在。抗原是由真菌和细菌在燃料上生长过程中产生的活细胞和代谢产物产生的。因此，抗原的存在是燃料系统中微生物污染的指标。抗原与非生物学来源的物质无关。 5.3.1 通过这种测试方法检测到的一些抗原在用杀生物剂处理后可以持续存在。看见 11.4 。 5.4 该测试方法是半定量的，可用于确定从燃料箱和系统中提取的样本中的污染是否可以忽略不计，或者是否存在中度或重度污染。 5.4.1 关于使用测试评估生物退化风险的更多信息，请参阅 附录X1 。 5.5 这些水平对操作员的重要性将取决于燃料类型、采样位置、采样的设备或设施以及特定的操作环境。 5.6 有关测试结果解释的进一步指南，请参阅指南 D6469 ，能源研究所关于调查石油燃料中微生物含量的指南，以及国际航空运输协会关于飞机油箱中微生物污染的指导材料。 5.7 有关取样的进一步指导，请参阅《实践》 D7464 。 5.8 测试可以在常规基础上进行，也可以用于调查事件。 5.9 微生物试验不用于确定是否符合燃料规范或限制。 燃料中微生物污染的规格限制的实施通常是不合适的，微生物污染水平不能单独或直接用于推断燃料质量或适用性。 5.10 在解释结果时，必须意识到，测试结果仅适用于测试的特定样品和样本，而不一定适用于散装燃料。微生物污染通常在燃料系统中表现出高度不均匀的分布，因此，对单个样本的分析很少能对存在的总体污染水平进行完整评估。 5.11 水相通常比燃料相含有高得多的微生物污染，因此，需要对结果进行不同的解释。 这就是为什么该测试方法报告水每毫升和燃料每升的抗原浓度。 5.12 此测试方法不同于其他一些方法（例如测试方法 D7687 和 D7978 )和实践（例如实践 D6974 )因为它可以在现场检测燃料或相关水样本中的微生物活性，并且不需要在实验室或无菌环境中进行。它可以在实验室中使用。 5.13 该测试方法不需要专业的微生物学经验或知识。 5.14 该测试方法提供了反映样本中总活性微生物污染的快速结果，并使结果能够在15 最小值。 5.15 本试验方法区分了 树脂H.resinae 、其他真菌和需氧细菌（参见 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 mm 2 s –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 <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. ====== 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 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 ).