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 Microtox®  

Microtox® Acute Toxicity testing is a bioassay testing system that can be used for screening wastewater effluents for acute toxicity. Microtox® is used in conjunction with current United States Environmental Protection Agency (USEPA) Whole EIffluent Toxicity test procedures. Microtox® can also be used for solid-phase toxicity testing of sediments and pure compound toxicity testing. The test method centers around the use of Vibrio fischeri (strain VRRL B-11177), a type of marine bacteria known to emit light due to an enzymatic reaction as part of cellular respiration. In Microtox® Acute Toxicity, this bioluminescence is measured by a photometer and is used as an indicator of toxicity. Once bacteria are exposed to toxic substances, cellular respiration is halted and the intensity of light emission by bacteria decreases. Thus, there is a negative correlation between bioluminescence and effect on bacteria, and light intensity can act as a measure of toxicity. Microtox® measures light emission of the bacteria under both exposed and unexposed conditions. Light intensity lost from exposed bacteria relative to that of unexposed bacteria is a measure of toxicity. Because bacteria are critical components of basic ecosystem functions, including nutrient and energy cycling, data from Microtox® Acute Toxicity tests can provide scientists with ecosystem health and potential toxicity. Microtox® data can be analyzed by calculating EC50 values based on concentration response curves.

Early History
With growing concern over environmental conditions in the United States in the 1970s, formation of the USEPA and passing of legislature like the Clean Water Act drove a demand for novel toxicity tests. Industries would now be required to follow more stringent regulatory framework, including the monitoring of their industrial activities. The petroleum industry in California initiated the search for a cheap and fast toxicity bioassay to monitor the drilling operations to replace traditional testing utilizing fish or invertebrates. Beckman Instrument Co in Carlsbad, California, was among the companies seeking to develop the new technique emphasizing speed, simplicity, standardization, and sensitivity. Headed by scientist Don Isenberg, who later wrote a paper detailing the development of Microtox®, the team at Beckman Instrument Co hoped to develop a method similar to a successful bacterial bioindicator of airborne chemicals which utilized luminescent bacteria. The hope was to find a strain which would elicit a measurable decrease in photochemical activity when exposed to contaminants that could be related to traditional aquatic toxicity tests.

Assay Development
Bacteria made an ideal option for researchers due to their quick response to environmental conditions, fast growth, and microscopic size. After inspiration from a paper by Johnsen et al. detailing the chemical processes related to biological reactions in bacteria, Beckman Instrument Co purchased more than 200 strains of luminescent bacteria to begin testing. The strain Vibrio fisheri was selected for its acute sensitivity. One final hurdle remained, as the team of scientists struggled to determine a method to effectively transport the bacteria for nationwide transportation. Beckman Instrument Co developed a proprietary method for reanimating bacteria after freeze drying it, and then engineered an instrument to measure the photochemical reaction while maintaining a specific temperature for the bacteria. With the conclusion of these final steps, the Microtox® assay was created.

Supplier History
The end product, along with the novel techniques for culturing the bacteria, were released in 1979 by Beckman Instrument Co. The Microtox® assay kit was seen in a side column in Environmental Science and Technology in 1980. The developers at eventually formed Microbics Corporation in in 1985, which was later changed to AZUR Environmental. Under AZUR, the Microtox® software was improved and MicrotoxOmni® was implemented for use with the Microtox® Model 500 Analyzer. In 2000, Strategic Diagnostics Incorporated purchased AZUR and continues to distribute Microtox® assay kits.

ASTM Method D 5660-96 and Microtox® Acute Toxicity Testing
The USEPA has adopted ASTM Method D 5660-96 (reapproved 2004) as their official procedure for use with Microtox® Acute Toxicity testing. This method calls for the use of standardized cultures of the marine bacterium Vibrio fischeri (reclassified from Photobacterium phosphoreum). A bioluminescent species, this bacterium emits blue-green light as the result of internal enzymatic activity.

Equipment
Equipment that is typically used for this method includes: a computer for data capture and analysis, a photometer and glass cuvettes, Absorbance Correction Cuvettes (ACCs), a temperature control device capable of maintaining 15 °C ± 0.5 °C and 5.5 °C ± 0.5 °C conditions simultaneously, 10 to 1000 μL and 1 to 5 mL pipettes, a timer, Microbial Reagent (freeze dried V. fischeri), Reconstitution Solution (non-toxic water), Sodium Chloride for use in preparation of 2.0 % NaCl and 3.4% NaCl diluents, Phenol and Zinc Sulfate Heptahydrate to act as reference toxicants, and pure water as defined by ASTM standards.

Test Organism
It is important to note that V. fischeri is a marine bacterium, and as such the salinity of some samples must be adjusted accordingly. Diluents of 2.0 % NaCl (for freshwater samples) and 3.4% NaCl (for marine samples) are typically used to accomplish this.

Sampling
Before testing can begin, a sample of wastewater effluent (or sediment) must be collected according to ASTM methodology (or equivalent standard). It is then necessary to derive control and treatment groups from this sample. Control groups are autoclaved and undergo alterations similar to that of treatment groups. At the end of the test, data from these two groups are then compared. The purpose of using control and treatment groups is to ensure that changes in toxicity are attributed to the treatment, and not to extraneous variables.

Aqueous Samples
Once the sample has been collected, it is prepared for testing. If a test sample is already in an aqueous state, then it is dispensed into one thermally controlled cuvette (maintained at 15 °C ± 0.5 °C). This cuvette is known as the primary sample. Nine additional cuvettes are then filled with the diluent appropriate to the sample type. The temperature of these cuvettes is maintained in a similar fashion to that of the primary sample. A serial dilution is preformed between the primary sample and the other cuvettes, which yield decreasing concentrations of both the sample and associated toxicants. An additional set of cuvettes are also prepared. These cuvettes are filled with the appropriate diluent and maintained at a steady temperature of 15 °C ± 0.5 °C. In addition to diluent, a reconstituted bacteria solution is added as well. The bacteria solution is created by mixing a frozen V. fischeri pellet with non-toxic pure water in a cuvette maintained at 5 °C ± 0.5 °C. These cuvettes are cycled through a photometer to produce initial luminescence readings. Once temperature has equalized in the first group of cuvettes, they are dispensed into the second group of cuvettes. The second group is the cycled through a photometer at intervals of 5, and 15, and 30 minutes. If toxicants are present within the sample, luminescence will decrease (enzymatic activity will cease). To obtain a direct measurement of toxicity, luminescence readings from the photometer are fed into Equation 1 and Equation 2, and are converted to gamma values (Γ). These values can then be used to determine an IC20 or IC50 (equivalent to an EC20 or EC50).

Sediment Samples
Sediment sample toxicity can also be measured, though samples must undergo additional preparation before toxicity testing can begin. To prepare a wet sediment sample, pore water within the sample is separated from the sediment by centrifuge. This pore water is decanted away leaving only a pellet of solids behind. A sample from this pellet is homogenized, dried, and weighed to obtain a dry weight. Diluent appropriate to the sample type is then added to a secondary representative sample, mixed, and then centrifuged again to separate pore water and sediment once more. However, in this instance the pore water is kept. If present, suspended solids are removed via positive pressure filtration and parameters including salinity, dissolved oxygen, conductivity, and pH are measured. Finally, the salinity of the sample is adjusted to reflect the sample type. Once these steps have been completed, the sample is ready to be tested in a manner similar to that of aqueous samples.

Sample Coloration and Accuracy
The accuracy this toxicity test can be adversely affected by a high degree of sample coloration. However, this can be corrected for with data reduction. To accomplish this, a small quantity of diluent appropriate to the sample is placed into a two chambered Absorbance Correction Cuvette (ACC). Diluent is then dispensed into one normal cuvette, while sample is dispensed into two other cuvettes. The samples and the diluent are allowed to reach thermal equilibrium with their respective cuvettes, and then reconstituted bacteria solution is added to the cuvette containing the diluent. The resulting solution is then mixed into both chambers of the ACC. A photometer is used to record luminescence data of the bacteria in the ACC for a short period of time. Then, the solution in the outer chamber of the ACC is emptied and replaced with sample from one of the two available cuvettes. Data pertaining to luminescence are once again recorded by the photometer for a short period of time. The data pertaining to the last several minutes of reading is used to reduce data from the actual toxicity test, thus accounting for the sample's high degree of coloration.

Baseline Toxicity
Baseline toxicity can also be determined for either type of sample by using an expedited version of the test that ignores many of the wait times associated with the procedure. If light output loss greater than 20 % over the first several minutes after sample addition, then the sample is diluted tenfold and the test is restarted. The endpoint of this range finding test would be when light output loss is less than 20 % after the first couple minutes after sample addition.

Equations
Equation 1. This equation is used to convert photometer measurements to gamma values. This is equivalent to an IC20 (EC20) or an IC50 (or EC50).

Γ(t)=(Io-It)/It

Γ(t) = gamma value at time t Io = initial light reading It = light reading at time t

Equation 2. Used to normalize Equation 1 by accounting for the addition of blanks to the test procedure. Factored into Equation 1 by Equation 3.

R(t)=(I(t)b)/(I(o)b)

R(t) = blank ratio for time t I(t)b = blank light reading at time t I(o)b = initial blank light reading

Equation 3. Similar to Equation 1, however with the blank ratio incorporated.

Γ(t)=(R(t)[Io-It])/It

Γ(t) = gamma value at time t R(t) = blank ratio for time t Io = initial light reading It = light reading at time t

Other standards similar to ASTM Method D 5660-96 include: EPS 1/RM/42 (Environmental Canada) and DIN 38412-37 (Deutsches Institut für Normung).

The Microtox® Acute Toxicity Test Kit
Microtox® Acute Toxicity testing builds upon the ASTM method. Though the testing process remains virtually the same, the Microtox® Acute Toxicity test kit offers the user both specialized equipment and software tailor-made for toxicity tests with V. fischeri. This allows the user to conduct tests in a manner that is quick and efficient. Such equipment includes: The Microtox® Model 500 Analyzer, with 30 wells capable of incubating samples at 15 °C ± 0.1 °C, MicrotoxOmni Software, which allows for the easy capture, analysis, and visualization of test data, and pre-made solutions (though these can easily be made within a lab).

Advantages
Microtox® Acute Toxicity testing could be an efficient means of screening effluent for toxicants in conjunction with current WET standards. The test utilizes roughly one million bacteria, negating the effect of undesired responses from small numbers of organisms on the credibility of the test. Additionally, each test requires only a small amount of sample, and the test is highly sensitive to sulfur and sulfur related compounds. The use of V. fischeri in the test has been backed extensively by over 350 peer-reviews scientific papers, and is thought to account for an ecologically relevant trophic level; bacteria are critical to energy and nutrient cycling within ecosystems. The Microtox® Acute Toxicity test is relatively cheap, with each test costing between $50 and $150 USD.

Disadvantages
The application of V. fischeri is considered to be somewhat limited, as the species is a saltwater bacterium. As such the salinity of freshwater test samples must be adjusted. This has the potential to alter toxicant properties, and may render samples as unrepresentative of actual effluent.

General Uses
Microtox® has been used worldwide for toxicity screening of chemicals and effluents, water quality and sediment contamination surveys, and environmental risk assessment. Microtox® has effectively been used in toxicity monitoring and assessment of industrial effluent, domestic wastewaters sewage, lake and river waters, agricultural runoff, leachates and aqueous extracts of soils and sediments,  groundwater, and drilling muds. Individual toxicity tests with organics, heavy metals, herbicides, and pesticides have also been successfully implemented. The test data generated by Microtox® has been used to compare with and predict outcomes of other bioassays. Microtox® test templates exist for acute, chronic, genotoxicity tests as well as for solid phase, pure compounds and aqueous extract. Microtox® is designed to be a benchtop instrument and not tested in a non-laboratory setting. The Company also markets a portable version of the Microtox® technology known as Deltatox®.

Regulations and Standards
The robustness of the Microtox® bioassays have resulted in its incorporation into multiple standards and regulations; Microtox® Acute Toxicity Test is a worldwide standard in environmental toxicology. Over 60 countries use the Microtox® as a regulatory tool, including the United Kingdom, France, Germany, Sweden, Netherlands, Canada, Italy, Spain, and Mexico. Standardized methods for Microtox® bioassays exist in Environment Canada (EC) 1992, Standard Methods for the Examination of Water and Wastewater, 1995, part 8050, American Society for Testing and Materials, 1995, D-5660-95 (ASTMD5660) the United States Public Health Service. and the International Organization for Standardization (ISO) standard methods, ISO 11348-3.

EPA Status
While the USEPA has adopted Microtox® as a resource for numerous studies and assessments USEPA do not regulate based on Microtox® for NPDES permits in the U.S. and its use in TIEs is limited. . The USEPA is in the process of further reviewing the appropriateness of Microtox® testing for potential use in regulation of discharge situations. Microtox® has been proposed for Whole Effluent Toxicity applications. The proposal offers two options for the incorporation of Microtox® testing. First, to use as a screening procedure implemented for discharges to marine and estuarine water. Secondly, to be used to establish NPDES permit limits as an additional phylogenetic group for which to evaluate a sample’s toxicity. Microtox® is not currently approved for use as a WET method by USEPA, but can be used by individuals to help assure compliance with wastewater treatment effluent permits like NPDES. The bacteria test results are sensitive to certain toxicants, but insensitive to others that can be toxic to most other aquatic organisms at similar concentrations.

Documented Correlations
Compiled data from Vibrio fisheri toxicity tests have demonstrated excellent correlation between many other common test organisms. The fathead minnow has one of the largest aquatic test data sets for more than 800 chemicals, and has been shown to have strong correlation with Microtox® results sufficient for predication of LC50s/EC50s within an order of magnitude. Other fish species include, but are not limited to, fathead minnow (Pimephales promelas), catfish (Ictalurus punctatus), goldfish (Carassius auratus), rainbow trout (Oncorhynchus mykiss), sheepshead minnow (Cyprinodon variegatus), and zebrafish (Brachydanio rerio). Other aquatic organisms have also shown consistent relationships such as water fleas (Daphnia magna), alga (Chlorella pyrenoidosa), crustaceans (Artemia salina), and ciliates (Tetrahymena pyriformis). . Among comparative toxicity tests using algae, crustaceans, rotifers, bacteria, and protozoan, the algae and bioluminescent bacterial assays demonstrated the highest level of sensitivity.

Potential Weaknesses
While Microtox® is widely applicable for screening a broad range of contaminants some exceptions have been observed. Microtox® is particularly insensitive to ammonia. Studies have suggested that it is less sensitive than other bioassays to metals as well.