In addition to the chemical, elemental, and physical tests described so far in this book, some miscellaneous tests are applicable to bio- fuels that may be considered environmental or ecological in nature.
One group of such tests is meant to estimate biodegradability and renewable content of test fuels or microbial contamination. Biofuel specifications usually do not contain specific biodegradation or renewable content limits per se. Over the last two decades, numer- ous standard testing methods and computer-based predictive models have been developed to help assess the environmental per- sistence, biodegradation, and toxicity of materials. Most of these methods have been developed for simple, soluble, and nonvolatile substances. In contrast, most fuels and lubricant products are not defined as such but rather are mixtures (simple and complex) of organic molecules, often relatively insoluble in water or volatile (or both). Also, additives can introduce metals and other inorganic compounds into the mixture. Nevertheless, the same standard methods were and are used to test these products. This often pro- duces misleading results and interpretations. Characteristics such as water solubility, vapor pressure, dissociation constant, and sorp- tion can render a specific test method inappropriate for many of these materials. In many cases, the results of different test methods for the same basic characteristics (e.g., biodegradation) cannot be directly compared [1].
Traditional lubricants used in machinery are usually toxic and are not readily biodegradable due to the poor biodegradability of their base oils. To preserve environmental and ground water sys- tems, there has been an increasing interest in using environmentally acceptable (EA) lubricants in environmentally sensitive industries such as construction, forestry, and agriculture. These EA lubricants are less toxic and readily biodegradable products. Currently, many EA lubricants have been formulated with renewable oils such as rapeseed, sunflower, corn, soybean, canola, and synthetic esters [2].
The biodegradability of lubricants allows them to break down in the environment, reducing negative effects from leaks and spills.
They can be nontoxic, meaning they will not harm operators, ani- mals, or plants that come in contact with the fluid. Furthermore, they are renewable. One of the main reasons for concern about lubricants’ environmental safety is that many industrial lubricants find their way into the environment. The National Oceanic and Atmospheric Administration estimates that more than 700 mil- lion gal of petroleum oil enter the environment each year—more
than half of which is released through irresponsible and illegal disposal.
Biodegradation
Biodegradation is a natural process caused by the action of microor- ganisms in the presence of oxygen, nitrogen, phosphorus, and trace minerals. Organic pollutants can support microbial growth and are converted into a series of oxidation products that ultimately end with carbon dioxide and water. Inherent biodegradability of a lubri- cant depends to a large extent upon its molecular structure and composition [2]. Typically, straight-chain aliphatic compounds (i.e., alkanes) are easily degraded; however, aromatic com pounds are degraded very slowly and are toxic. Polymeric materials are among the most resistant to microbial attack. Oils derived from renewable resources are more biodegradable than petroleum-based oils. In addition, the water solubility (or dispersibility) of lubricant prod- ucts, their molecular size, pH of the solution, types of materials, temperature, total dissolved solids, and their toxicity all affect their biodegradability. Among these, it is strongly believed that molecu- lar structure and toxicity may play the most significant role in the biodegradation of lubricants [2]. Some terms used in this context are explained in Table 6.1.
Biodegradation is the biologically mediated transformation of a material. The microorganisms that accomplish the biodegrada- tion are very important to the nature of the result. Generally, the interest in studying biodegradation is for using the results to pre- dict the environmental fate of a chemical. Thus, the microorgan- isms used in laboratory tests are mixtures of species collected from particular environments. For a variety of reasons, sewage sludge has become the standard source of mixed inoculum for introduc- tion into laboratory biodegradation test systems.
In the aquatic environment, bacterial numbers are lower than in sewage, and there are also low concentrations of a variety of chemicals and nutrients. In standardized testing, the organisms are constrained within a small volume and are presented with a high concentration of the test material as the sole source of carbon.
Those organisms capable of degrading the chemical will use it as a source of carbon for new cells and energy. Pre-exposure to petro- leum products can result in a 1 % to 10 % increase in the total pop- ulation of petroleum-degrading microorganisms. The rate of this
DOI: 10.1520/MNL772015001106
biodegradation reflects the growth rates of those particular organ- isms under the conditions of the test. The test results are used to classify the persistence of the material and to estimate its half-life in the environment [1].
Certain environmental conditions (e.g., availability of oxy- gen) greatly influence the types of organisms and their metabolic pathways. Standardized tests exist for aerobic and anaerobic condi- tions in the major environmental compartments (freshwater, marine water, sediments, and soils). In some cases, simulation tests are intended to replicate the natural conditions of the organisms, their environment, and their exposure to the chemical [3]. The main terms used to describe biodegradation—primary and ulti- mate—distinguish between two extents of biodegradation.
“Primary” refers to the initial transformation from the parent material, and “ultimate” refers to mineralization of the material.
The rate is described by the terms inherent and ready. In general, to be classified as “inherently” biodegradable, there must be unequiv- ocal evidence of biodegradation by any test method. The Orga- nization for Economic Cooperation and Development (OECD) methods require 20 % degradation for this classification. “Ready”
biodegradability is a regulatory classification originating in the European Union that has very specific criteria. See Hinman for further discussion of this classification [1].
There are two commonly used designations for biodegrad- ability: “readily” and “inherently.” Readily biodegradable is defined as breaking down very rapidly in the environment by a prescribed amount in a specific time frame. Inherently biode- gradable is misleading to the unknowing because it means the substance has the propensity to break down with no defined amount or time frame. Thus, an inherently biodegradable prod- uct breaks down very slowly over time, usually in terms of years.
These type of products can persist in the environment for several years, continuing to cause substantial damage. They require long-term remediation due to their environmental persistence.
Typically, these products are petroleum-based, such as conven- tional lubricants [4].
Certain standardized tests have been developed for regula- tory purposes to evaluate the rate and extent of biodegradation. See Table 6.2 [5]. A number of biodegradation tests are available and differ mainly in the method of analysis. The different assays give different results. The analytical methods dictate details of the pro- cedures and result in different applicabilities for each test.
Generally, the disappearance of dissolved organic carbon is more rapid than the utilization of oxygen or the production of carbon dioxide. Standard tests for biodegradation have been issued by several organizations such as ASTM, OECD, the European Table 6.1 Terminology Used in Biodegradation Tests
Term explanation Standard
Aerobic Taking place in the presence of oxygen. ASTM D6006
Anaerobic Taking place in the absence of oxygen. ASTM D6006
Biodegradation The transformation of a material resulting from the complex enzymatic action of microorganisms (e.g., bacteria, fungi). It usually leads to the disappearance of the parent structure and to the formation of smaller chemical species, some of which are used for cell metabolism. Although typically used in reference to microbial activity, it may also refer to general metabolic breakdown of a substance by any living organism.
Inherent
biodegradability Classification of chemicals for which there is unequivocal evidence of biodegradation (primary or ultimate) in any test of biodegradability.
Inoculum Living spores, bacteria, single-celled organisms, or other live materials that are introduced into a test medium. ASTM D6384 Primary
biodegradation
Degradation of the test substance resulting in a change in its physical or chemical properties, or in both. ASTM D6006 Ultimate
biodegradation Degradation achieved when a substance is totally utilized by microorganisms resulting in the production of carbon dioxide (and possibly methane in the case of anaerobic biodegradation), water, inorganic compounds, and new microbial cellular constituents (biomass or secretions, or both).
ASTM D6006
Table 6.2 Examples of Biodegradation Tests
attribute Categories Measurement Properties
Extent of biodegradation Ultimate Oxygen, carbon dioxide, methane Measures total conversion to inorganic forms; mineralization
Primary Specific analysis Based on analysis of specific chemical or chemical class; abiotic losses controlled Removal Specific analysis Primary biodegradation; both abiotic and biodegradation
Ease of biodegradation Ready Oxygen, carbon dioxide, dissolved organic carbon (DOC)
Regulatory definition of “rapid” biodegradation; uses <30 mg/L nonadapted inoculum;
reaches minimum 60 % degradation to oxygen, carbon dioxide, or 70 % removal of DOC in 28 days; must go from 10 % to the pass level (60 % or 70 %) in 10 days Inherent Oxygen, carbon dioxide,
methane, DOC, specific analysis Enhanced conditions to show possibility of eventually biodegrading; generally a high biomass adapted inoculum
Simulation Specific analysis Reflects actual environmental behavior; difficult to simulate most exposure situations From Ref. [5].
Commission (EC), the International Organization for Standardization (ISO), and the U.S. Environmental Protection Agency (EPA). Hinman has provided an extensive list of com- monly used standardized tests, the general characteristics of the tests, and their general applicability [5]. A summary of these tests is given in Table 6.3 [5]. In the United States, government authoriza- tion for the manufacture of new chemicals is regulated under the Toxic Substances Control Act (TSCA), which requires a premanu- facturing notification be submitted for EPA review before full- scale manufacture of the material. In Canada, the New Substances Provisions of the Canadian Environmental Protection Act per- forms an equivalent role. In the European Union, Council Directive 67/548/EEC-Classification, Packaging, and Labeling of Dangerous Substances drives the premarketing notification process. Of the many tests required for these notifications, those for biodegrada- tion testing are listed in Table 6.4 [5].
Comparing the results from different test methods for the same or similar materials can be problematic. The “percent biodeg- radation” is very dependent upon what type of biodegradability was tested. As a result of this and various complications resulting from the use of inappropriate tests for the physical characteristics of the material, the comparison of biodegradability among materi- als is only quantitative if they are tested in the same test systems using the same inoculum prepared at the same time. The major use of biodegradation tests is to provide an estimate of the potential of a material to degrade in the environment [1].
In general, hydrocarbons are biodegradable in varying degrees. Fuels and lubricants are mixtures with some components having low water solubility that often limits the concentration in solution and thus their availability to the microorganisms. The aromatics tend to be slightly more water-soluble and give “better”
results in standardized tests than the paraffins. Among the paraf- fins, the linear hydrocarbons are considered more biodegradable than the branched hydrocarbons. This results from microbial oxi- dation of one end of the linear molecule to a carboxylic acid. The metabolic processes in nearly all organisms can easily degrade the resulting fatty acid. The presence of branching hinders degrada- tion to some extent [5].
A partial list of available ASTM biodegradation tests for vari- ous classes of petroleum products and lubricants is given in Table 6.5. The EPA tests for biodegradability are given in Table 6.6. At present, there are no published ASTM or other tests for biodegra- dation for biofuels. Of the ASTM test methods listed in Table 6.5, at least three are applicable to lubricant and grease testing. These test methods measure the ultimate biodegradation of the lubricants.
The evolved carbon dioxide (CO2) methods work by collecting the evolved CO2 from the biological breakdown of the lubricant. At the completion of the test, the CO2 evolved is compared to the theoret- ical CO2 that can be produced from the biological breakdown and reported as the percentage of biodegradation. ASTM D6731 works by collecting the oxygen consumed from the biological breakdown of the lubricant. The oxygen consumed is compared to the amount Table 6.3 Examples of Relevant Biodegradation Test Methods
Test Standard /Reference environment biodegradation
extent biodegradation
Rate
DOC Die-Away OECD 301 A; EC C-4A; ISO 7827 Aquatic Ultimate Primary Ready
Modified Sturm Method OECD 301 B; EC C4-A; ASTM D5864; ISO 9439; EPA 835.3110 Aquatic Ultimate Ready
Gledhill Method ASTM D6139 Aquatic Ultimate Ready
MITI (I and II) OECD 301 C; EC C4-F(I); OECD 302 C(II); EPA 835.3110 Aquatic Ultimate Ready
Closed Bottle OECD 301 D; EC C4-D; ASTM e1720; ISO 10707; EPA 835.3110 Aquatic Ultimate Ready
2-Phase Closed Bottle ISO 10708 Aquatic Ultimate Ready
Modified Screening OECD 301 E; EC C4-B; ISO 7827 Aquatic Ultimate Ready
Respirometry OECD 301 F; EC C4-D; ISO 9408; EPA 835.3110 Aquatic Ultimate Primary Ready Inherent
Sea Water OECD 306 Aquatic–Marine Ultimate Inherent
Zahn-Wellens/EMPA OECD 302 B; ISO 9888; EPA 835.3200 Aquatic Ultimate Primary Inherent
Sludge–Semicontinuous (Mod. semi- continuous activated sludge [SCAS])
OECD 302 A; ASTM e1625; ISO 9887; EPA 835.3210; EPA 835.5045
Aquatic Ultimate Primary Removal
Inherent
Sludge – Continuous (Coupled Units) OECD 303 A; ISO 11733 Aquatic Ultimate Simulation Inherent
Soil OECD 304 A; ISO 14239; EPA 835.3300 Soil Ultimate Simulation Inherent
Biogas Generation–Anaerobic ECETOC TR 28; ISO 11734 Aquatic Ultimate Primary Inherent
2-Stroke Oil CEC L-35-A-94 Aquatic Primary Inherent
CO2 Generation in Sealed Vessels ISO 14593; CONCAWE Rpt. No. 99/59; Battersby 1997;
EPA 835.3120
Aquatic Ultimate Inherent
Porous Pot Test ASTM e1798 Aquatic Ultimate Simulation Inherent
Shake-Flask Die-Away Test ASTM e1279; EPA 835.3170+ Aquatic–
Surface Water Primary Inherent
From Ref. [5].
Table 6.4 Classification of Biodegradation Tests
Characteristics USa-TSCa european Union Canada
Ready biodegradation 835.3110 OECD 301 OECD 301
OECD 310 OECD 310
OECD 311 OECD 311
Inherent biodegradation 835.3100 OECD 302 OECD 302
835.3120 OECD 304 OECD 304
835.3200 835.3400 835.5045
Simulation biodegradation OECD 303
OECD 309 From Ref. [5].
Table 6.5 International Biodegradation Tests for Petroleum Products and Lubricants
aSTM ISO OeCD
ASTM D5846, Biodegradability by Modified Sturm Test ASTM D5864, Aerobic Aquatic Biodegradation of
Lubricants or Their Components ISO 7827, Aqueous Medium of the “Ultimate” Aerobic Biodegradability of Organic Compounds—Dissolved Organic Carbon
OECD 301, Ready Biodegradability OECD 301B, CO2 Evolution Test (the Modified Sturm Test)
OECD 301C, Modified MITI Test ASTM D6006, Guide for Assessing Biodegradability of
Hydraulic Fluids ISO 9408, Ultimate Aerobic Biodegradability of Organic
Compounds in Aqueous Medium OECD 302, Inherent Biodegradability
ASTM D6139, Aerobic Aquatic Biodegradation of
Lubricants or Their Components ISO 9439, Ultimate Aerobic Biodegradability of Organic
Compounds in Aqueous Medium OECD 304, Inherent Biodegradability in
Soil ASTM D6384, Terminology Relating to Biodegradability
and Ecotoxicity of Lubricants ISO 9887, Aerobic Biodegradability of Organic Compounds in an
Aqueous Medium—SCAS Test OECD 306, Biodegradability in
Seawater ASTM D6731, Aerobic, Aquatic Biodegradability of
Lubricants or Components in a Closed Respirometer ISO 9888, Ultimate Aerobic Biodegradability of Organic
Compounds in Aqueous Medium—Static Test OECD 309, Aerobic Mineralization in Surface Water—Simulation Test ASTM D6866, Renewable Content of Samples Using
Radiochemical Analysis
ASTM e1279, Biodegradation by a Shake-Flask Die-Away Method
ISO 10634, Preparation and Treatment of Poorly Water- Soluble Organic Compounds for Biodegradability in an Aqueous Medium
OECD 310, Ready Biodegradability—CO2 in Sealed Vessels
ASTM e1625, Biodegradability of Organic Chemicals in
Semi-Continuous Activated Sludge (SCAS) ISO 10707, Ultimate Aerobic Biodegradability of Organic
Compounds in an Aqueous Medium by Closed Bottle Test OECD 311, Ready Anaerobic Biodegradability in Diluted Anaerobic Sewage Sludge
ASTM e1720, Ready and Ultimate Biodegradability of Organic Chemicals in a Sealed Vessel CO2 Production Test
ISO 10708, Ultimate Aerobic Biodegradability of Organic Compounds in an Aqueous Medium in a Two-Phase Closed Bottle Test
ASTM e1798, Assessing Treatability or Biodegradability,
or Both, of Organic Chemicals in Porous Pots ISO 11266, Guidance for Laboratory Testing for Biodegradation of Organic Chemicals in Soil Under Aerobic Conditions
ASTM e2170, Anaerobic Biodegradation Potential of Organic Chemicals Under Methanogenic Conditions
ISO 11733, Elimination and Biodegradability of Organic Compounds in an Aqueous Medium (Activated Sludge Simulation Test [ASST])
ISO 11734, Ultimate Anaerobic Biodegradability of Organic Compounds in Digested Sludge
ISO 14593, Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium
ISO/TR 15462, Selection of Tests for Biodegradability ISO 16221, Determination of Biodegradability in the Marine Environment
of the theoretical oxygen that should be consumed by the break- down or the oxidation of the test lubricant. The amount collected is reported as a percentage of the total biodegradation. For all of these methods, it is important to obtain accurate elemental analysis of the test material in order to have a relevant test result [6].
The ASTM test methods are very similar to each other and simulate the biodegradation process used in a waste treatment facility. But they have a long testing time (28 days). Additionally, these tests also have very poor precision due to the various and multiple inoculum sources. A problem with inoculums is that although one may get it from the same source, differences in the microorganism population can vary from batch to batch [2].
In-Sik Rhee has developed a new way to determine biode- gradability of lubricants using a biokinetic model [7]. Currently available ASTM and OECD test methods take a long time (28 days), and special biological knowledge is required. To resolve this problem, a biokinetic model was developed based on the composi- tion of lubricants. The advantage of this biokinetic model is that it can predict the biodegradability of lubricants within a day without the use of microorganisms. The new test method (ASTM D7373) has excellent correlation with the aerobic closed respirometer test, ASTM D6731.
ASTM D5846, Universal Oxidation Test for Hydraulic and Turbine Oils Using the Universal Oxidation Test Apparatus
This test was developed for evaluating the oxidation stability of petroleum-based hydraulic oils and oils for steam and gas turbines.
It has also been used to evaluate the oxidation stability of fluids made with synthetic base stock and in-service oils.
Significance
Degradation of hydraulic fluids and turbine oils, caused by oxida- tion or thermal breakdown, can result in the formation of acids or insoluble solids and render the oil unfit for further use. This test method can be used to estimate the relative oxidation stability of petroleum-based oils. The correlation between results of this test
and the oxidation stability in use can vary markedly with service conditions and with various oils.
analySiS
An oil sample is contacted with air at 135°C in the presence of cop- per and iron metals. The acid number and spot-forming tendency of the oil are measured daily. The test is terminated when the oxi- dation life of the oil has been reached. The oil is considered to be degraded when either its acid number (measured by test methods ASTM D974 or ASTM D664) has increased by 0.5 mg KOH/g over that of new oil or when the oil begins to form insoluble solids so that when a drop of oil is placed onto a filter paper it shows a clearly defined dark spot surrounded by a ring of clear oil.
PreciSion
Based on two interlaboratory studies, the following precisions have been obtained for antiwear hydraulic oils as well as steam and gas turbine oils. No information is available for biofuels or biolubricants.
ASTM D5864, Aerobic Aquatic Biodegradation of Lubricants or Their Compounds
This test method is a version of the OECD 301 B Modified Sturm Test that closely simulates wastewater biodegradation conditions.
It was designed to determine the degree of aerobic aquatic biodeg- radation of lubricants upon exposure to inoculum under labora- tory conditions. In this test, the biodegradability of a lubricant is expressed as a percentage of maximum carbon conversion under well-controlled conditions for a period of 28 days.
Significance
This test method covers the determination of the degree of aerobic aquatic biodegradation of fully formulated lubricants or their com- ponents upon exposure to an inoculum under laboratory condi- tions. It is intended to specifically address the difficulties associated with testing water-insoluble materials and the complex mixtures found in many lubricants. This test method is designed to be appli- cable to all lubricants that are not volatile and are not inhibitory at the test concentration of the organisms present in the inoculum.
ScoPe
In this test method, the degree of aquatic biodegradation of a lubricant or components of a lubricant is measured by the amount of evolved carbon dioxide upon exposure of the test material to an inoculum. The plateau level of carbon dioxide evolution in this test method will suggest the degree of biodegradability of the lubricant. Test substances that achieve a high degree of
Oils Repeatability Reproducibility
Antiwear hydraulic oils 0.0614 X 0.0918 X
Steam and gas turbine oils 0.0486 X 0.1400 X Note: Where X is the average of two results.
Table 6.6 U.S. EPA Biodegradability Tests for Petroleum Products and Lubricants
US ePa Subject
835.3100 Aerobic Aquatic Biodegradation 835.3110 Ready Biodegradability
835.3120 Sealed-Vessel Carbon Dioxide Production Test 835.3170 Shake-Flask Die-Away Test
835.3180 Sediment/Water Microcosm Biodegradation Test 835.3210 Modified SCAS Test
835.3300 Soil Biodegradation
835.3400 Anaerobic Biodegradability of Organic Chemicals