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For more than two decades, an alternative toxicity screening method has been available that when compared to traditional toxicity testing protocols offers the following benefits:


1. Compact – Eight to 10 feet of bench-top space and some room in the lab freezer for reagents, rather than entire laboratories and animal containment facilities


2. Fast – Results returned in minutes rather than days or months


3. Economical – A few hundred dollars compared to multiple thousands to run a test


4. Easy to perform – A person skilled with reasonable laboratory techniques who can operate a disposable micropipette and make serial dilutions will be an expert within days


5. Reliable – Excellent repeatability and reproducibility, easy quality control checks


The alternative toxicity screening method is known as the Microtox®


Rapid Toxicity Testing System. Developed in the late


1970s and commercialised by the Microbics Corporation, the ownership of the technology has gone through acquisitions and mergers since its development. Currently, it is owned by Modern Water (www.modernwater.com) in Guildford, United Kingdom.


The Microtox test uses bioluminescent bacteria as its test species. The simplicity of the test is that a measurable activity of the living luminescent bacteria is the production of light. If the bacteria’s metabolism slows or the bacteria die, the quantity of light lessens or ceases. The Microtox test measures a 50 percent effective concentration (EC50) rather than the traditional LD50. The EC50 is the concentration of a sample at which a 50 percent reduction in bioluminescence occurs and it is analogous to the 50 percent inhibition concentration (IC50) used in other toxicity tests.


The Microtox testing system provides the user with the tools necessary to quickly prepare the bacteria, quantify its light production, serial dilute the product to be tested, incubate, re-measure the light production, collect and analyse the data, and produce an EC50 value.


While the Microtox system is easy to use and cost-effective, it also has some obstacles to overcome when evaluating lubricants. Like other toxicity protocols used for evaluating lubricants, there is the aqueous/nonaqueous dilemma. The luminescent bacteria used in the Microtox protocol needs a pH-controlled saline solution (the laboratory equivalent of seawater) to be activated from a freeze-dried long-term storage state. Adding the lubricant directly to the bacteria in the saline solution does not produce useable data.


Spilled Lubricant To work around the dilemma, a laboratory scale model of what would occur in the real world if there were an oil spill or accidental release of a lubricant into a body of water is produced. In reality, spilled lubricant will float to the water’s surface and form a two-phase solution of water with an oil slick on top. Wind, sunlight, wave action and currents will stir and mix the two phases until the lubricant biodegrades, is physically removed from the water or the spilled lubricant becomes so dissipated it is no longer detectable.


Regardless of how the spilled lubricant is removed from the water, while it is present, there is potential for the water to extract toxic material from the oil into the water phase. This extraction process can be duplicated in the laboratory. Using a known quantity of the pH-controlled saline solution to act as the water phase, a known quantity of the lubricant to be tested is added to the saline solution. This two-phase solution is placed in a laboratory shaker and agitated to duplicate water currents and wave action. Upon completion of agitation, the two-phase solution is separated and the water phase is tested in the Microtox protocol. No formal procedure exists for the extraction process; however, ASTM D6081 “Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation” is an excellent reference and a good guideline to follow. Trial and error will dictate how long and how hard the solution is shaken, with the goal being a uniform and consistent extraction, making for more reproducible final data.


Limitations Like all toxicity tests, there are limitations to the bacteria used in the Microtox test. In technical literature, it has been reported that the bacteria are less sensitive to some common contaminants (some metals and ammonia compounds) than other traditionally accepted toxicity protocols. Conversely, the bacteria are more sensitive to sulphur and sulphur compounds. Simply put, the Microtox method results do not correspond one-to-one with the results produced by the other traditional toxicity methods, however a high degree of correlation exists. This does not make the Microtox results right or wrong; it merely requires more consideration and evaluation of the results.


A distinct advantage of the utilisation of bacteria as a test organism over other species is the population of bacteria utilised in a test verses other conventional species. In the Microtox protocol, up to one million bacteria are utilised per test vial, while other protocol use significantly less.


The Microtox method results are not influenced by the responses of a small number of test organisms. An additional advantage is that bacteria are about as far down the food chain as anything can get. If a test substance can be toxic to a very low form of life, how far up the food chain does it remain toxic? Also, how does the test substance ultimately affect the food chain?


The Microtox protocol has been slowly but steadily gaining acceptance as a screening tool for toxicity evaluation. Worldwide, there has been a faster and more general acceptance than in the United States. Methods and procedures that are well documented in more than 350 peer-reviewed papers have led to “standards status” in France, Germany, Netherlands, Spain, Sweden, United Kingdom and other countries. Additionally in Canada, the Energy and Utilities Board has established a method to estimate toxicity of oil well drilling sump fluids using a procedure based on the Microtox protocol.


In the United States, there is an ASTM method, ASTM D5660 “Standard Test Method of Assessing the Microbial Detoxification of Chemically Contaminated Water and Soil Using a Toxicity Test with a Luminescent Marine Bacterium.” The standard was developed in the mid-1990s, reviewed, and was published in 1996. More recently, the U.S.


Continued on page 14


12


LUBE MAGAZINE NO.141 OCTOBER 2017


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