James Giese

Dioxin Testing
Dioxin is an overall term used to describe a family of highly toxic and chemically related compounds. The formal name forB this family of chemicals is polychlorinated dibenzo-p-dioxin (PCDD). Although this group of compounds originally represented a group of about 17 chlorine-substituted chemicals, it has become, in recent years, moreB inclusive of other chemical families as well.

USDA Agricultural Research technician loads dioxin samples into a gas chromatograph/mass spectrometer (GC/MS) autoinjector for analysis.

Dioxins are released into the air from industrial processes, such as commercial or municipal waste incineration and from burning fuels, such as wood, coal, or oil. Burning of household trash and forest fires can also result in the release of dioxins and the related furans into the environment. Accidental or intentional release of transformer fluids also has resulted in the presence of dioxins in the environment.

Because environmental dioxins tend to accumulate in the fat of food-producing animals, consumption of animal-derived foods, such as meat, poultry, eggs, fish, and dairy products, is considered to be the major route of human exposure.

Dioxins are a concern because exposure may result in adverse health effects. Studies have suggested that high dioxin levels to which some industrial workers were exposed in the past led to reproductive and developmental problems, increased heart diseases, increased diabetes, and increased cancer. In 1999, the International Agency for Research on Cancer classified a dioxin compound—2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)—as a human carcinogen. TCDD has become an important standard for measuring overall toxicity.

EU and U.S. Regulation
Throughout 1999 and during the next few years, concerns about dioxin in the food supply grew, especially in the countries of the European Union. On July 1, 2002, the EU issued regulatory limits on dioxin in food and feed marketed in the EU. The limits stem from a November 2001 EU Council regulation (IP/02/1698) setting legally binding limits on the presence of dioxin in food. An equivalent Directive (IP/02/1670) setting maximum limits in animal feed was adopted at the same time. Any food, feed, or feed material exceeding these strict limits is excludedB from the food chain.

The EU regulations will affect United States exports of animal by-products, cereals, feed and feed ingredients, fish and fish products, milk and milk products, eggs and egg products, oils, fats, fruits, and vegetables. So U.S. exporters will need to monitor and analyze their products for dioxins.

Although the U.S. government does not currently regulate dioxin limits in foods, the Environmental Protection Agency and industry have worked together to reduce emissions from numerous combustion processes, resulting in a significant decline in levels of dioxins and furans in the U.S. For more information, see EPA's dioxin Web site at cfpub.epa.gov/ncea/cfm/dioxin.cfm. Because dioxins break down very slowly, dioxins released in the past from both manmade and natural sources still exist in the environment and cannot be quickly reduced.

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The Food and Drug Administration has been concerned about dioxins and has been monitoring specific foods with the goal of identifying ways to reduce dietary exposure. For example, since 1995, FDA has been monitoring dioxin levels in fish, shellfish, and dairy products. In 1999, FDA initiated dioxin analysis of foods collected under its Total Diet Study (TDS) survey. This annual survey of market baskets, representing 265 core foods (ready-to-eat) in the U.S. food supply, assesses levels of various pesticide residues, contaminants, and nutrients in foods and estimates intakes of these substances in representative diets of specific age-sex groups in the U.S.

In May 2002, the U.S. Dept. of Agriculture's Food Safety and Inspection Service announced that it was conducting a survey to gather information on dioxin in U.S. meat and poultry products. Data from the survey will be used to determine whether the amount of dioxin present in meat and poultry remains low and whether steps can be taken to reduce dioxin further. FSIS will use the information from the survey as it considers approaches to managing dioxin risks in the future. For more information, see www.fsis.usda.gov/oa/background/dioxins02.htm

In a speech last October, FDA Deputy Commissioner Lester Crawford asked the following questions about dioxins in foods: How can FDA manage risk when environmental levels are decreasing, and what scarce resources should be spent on surveillance?

According to FDA, because dioxin analysis is costly and time consuming, available data on background levels in most foods and feeds are limited. In fact, for many foods and most feeds, there are no data. Consequently, it is difficult to determine where and how dioxin levels in foods and feeds can be further reduced. FDA is expanding its monitoring program to obtain more comprehensive data on background levels, as well as to identify opportunities to reduce human exposure to dioxins.

Testing and Analysis
One analytical strategy for measuring dioxin and dioxin-related compounds is the accepted gas chromatographic-mass spectrometric (GC/MS) method, which measures individual compounds. The published EPA analytical method for analysis of dioxin and related compounds is Method 1613. Information on EPA analytical methods for dioxins and other contaminants is available at www.epa.gov/epahome/Standards.html.

By separately identifying each individual dioxin compound in a sample by the GC/MS method, individual concentrations are determined. Then the concentration of each compound is multiplied by its specific toxicity relative to TCDD. From the sum of the relative toxicities of all observed compounds, a weighted average is calculated to give a toxicity equivalency quotient (TEQ) for the entire mixture. Basically, TEQ is represented as TCDD equivalents; i.e., it representsan estimate of the maximum toxicityB of the sample. In practice, each compound is assigned a Toxicity Equivalency Factor (TEF) by EPA. This factor is multiplied by the measured concentration, and the sum of these values is the TEQ. It is expressed in the units of the analysis, generally as parts per trillion for a food sample. The final weighted average is intended to represent the aggregate toxicity of the whole sample. Although the GC/MS method allows investigation of individual dioxin compounds, it is often too costly or time-consuming for sample screening. In addition, according to some, it is not as reliable for predicting the toxicities of complex mixtures of the individual compounds. This is because the toxicity of individual compounds, when present in mixtures, is subject to complex interactions. Whole body toxicity responses often depend on many environmental factors, thus minimizing—and, to a considerable extent, making moot—the necessity for accurate measurement of individual congener concentrations.

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As an alternative to GC/MS analysis, efforts in recent years have been made to develop methods where the toxicity ofB the original unfractionated sample can be directly estimated and individual compounds do not have to be separated. Using this strategy, the TEQ or its equivalent of the entire mixture is measured. Several bioassay-based methods have been developed to address these needs. Some these methods depend on the maintenance and use of cell culture systems or radioactive components, both of which are relatively inconvenient and/or expensive.

According to one supplier of these methods, bioassays offer the ability to screen a large number of samples at a reasonable cost. The main requirement for such a method will be the precision of measuring aliquots of the same sample. It is necessary to know, for example, whether repeat assays of the same sample yield the same result. In statistical terms, the coefficient of variation should be less than about 20%.

When using these methods for screening, the main task is to identify excess levels of dioxin-like material in comparison with background. For those samples requiring further investigation, it may be useful to know something about the composition of dioxin-like material for purposes of research or understanding the effects of production and treatment. In this case, GC/MS analysis will likely be necessary to obtain pure compounds for study in isolation.

Screening Kits
Among the dioxin-screening kits on the market are the following:
Paracelsian, Inc. offers an immunoassay that makes use of a key biological pathway for toxicity that is used by all of the dioxins, furans, polycyclic aromatic hydrocarbons, and other related toxins. Research indicates that dioxins and related compounds initiate their toxic effects by binding to the aryl hydrocarbon (Ah) receptor proteins of cells. It is this transformed receptor protein, bound to the DNA, that enhances the synthesis of enzymes, which initiate a cascade of chemical events causing the toxic response.

Ah-I specifically targets and detects the transformed Ah-receptor complex using an optimized enzyme immunoassay in a simple ELISA microtiter-tray format. According to the company, its new bioassay-based methods possess the capacity to measure the diverse group of dioxin chemicals. Simultaneously measuring all chemicals capable of binding and transforming the AhR protein gives a more comprehensive estimate of potential toxicity.

The results obtained are expressed as "equivalents" of whichever reference material is used to develop the standard curve. Usually this curve is generated from TCDD, so the results would be "TCDD equivalents," or it could be equivalents in reference to a standard fly ash mixture. Whichever reference material is used, the company says that it must be remembered that the reference value itself is characterized not only by a definitive concentration but also by an arbitrary toxicity outcome.

Equipment required for the test is relatively low in cost and readily available. The assay is said to be simple to perform and sensitive to the respective toxicity of the dioxins and related compounds of interest. Toxicity is measured as the ability of the sample to transform the Ah-receptor. The result therefore denotes total toxicity from all toxins present in the sample. More information is available from Paracelsian, Inc., Langmuir Laboratories, 95 Brown Rd, #1005, Ithaca, NY 14850 (phone 888-689-4224; fax 607-257-2734).

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Cape Technologies, L.L.C. offers the DF1 High Performance Dioxin/Furan Immunoassay Kit. The kit is designed for screening analysis of polychlorinated dibenzo-dioxins and polychlorinated dibenzo-furans (PCDD/Fs) at preselected or user-determined levels.

During testing, PCDD/Fs are specifically bound by the anti-dioxin antibodies, which have been immobilized on the immunoassay's tube surface. After washing away the unbound material, the bound PCDD/Fs remain, and a competitor-HorseRadish Peroxidase (HRP) conjugate is added. Bound PCDD/Fs occupy the dioxin-binding sites of the antibodies in proportion to the PCDD/F content of the sample and prevent binding of the competitor-HRP conjugate. After a short incubation, unbound conjugate is removed and the test tubes are washed thoroughly. The amount of conjugate bound by the anti-dioxin antibody is inversely related to the amount of PCDD/Fs originally present in the sample.

Finally, a solution of chromogenic HRP substrate and hydrogen peroxide is added to the test tubes. Color developmentB is directly proportional to enzyme concentration and inversely related to the PCDD/F concentration in the original sample. The test tubes are analyzed using a tube reader or spectrophotometer to measure the optical density (OD) at 450 nm. The OD values of unknown samples are compared to those of standards to determine the level of PCDD/Fs in the samples.

The company offers applications notes for soil and fly ash, including immunoassay-specific sample preparation methods. Quantitative use of the kit is said to be possible for advanced users. More information is available from Cape Technologies, L.L.C., 3 Adams St., South Portland, MA 04106-1604 (phone 207-741-2995; fax 207-741-2996).

Triangle Laboratories has recently developed a screening assay, called RapidScreen, for selected food products, that will provide a yes-or-no answer for dioxin/furan TEQ at a predetermined value. The method is based on high-resolution mass spectrometry and is said to be as sensitive as the traditional analytical methods and as cost effective as bioassays.

The assay is based on use of a marker set of specific chlorinated dioxin and furan isomers that provides an estimate of the TEQ of a sample. The analytical technique employed is based on the principles of EPA methods 8290 and 1613, which utilize isotope dilution mass spectrometry. By using C13-labeled analogues as internal standards, the assay is claimed to not be subject to false negatives. In addition, by utilizing the selectivity of high-resolution mass spectrometry, the risks of false positives due to interfering compounds are reduced.

The sample results are generated in a short turnaround time of less than 5 days. Two-day turn-around is said to be available with appropriate prior planning. Traditional analyses often take 14– days for results to be available. More information can be obtained from Triangle Laboratories, Inc. 2445 S. Alston Ave., Durham, NC 27713 (phone 919-544-5729; fax 919-544-5491).

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Glassware Washer
, the Flask Scrubber, is an automated laboratory glassware cleaning system. The left-hand cabinet holds detergent and neutralizing acidB rinse dispensers. A pump feeds liquid detergent to the washer during the wash cycle, and another pump feeds neutralizing rinse to the washer during the rinse cycle. Separate alarms alert the user when levels are low. A HEPA filter removes particulates from the air introduced duringB the forced-air drying cycle. A water conductivity sensor measures water quality, which is displayed on the control panel. The sensor helps monitor the removal of contaminants and detergent carryover at the final rinse cycle. For more information, contact Labconco Corp., 8811 Prospect Ave., Kansas City, MO 64132-2696 (phone 816-333-8811; fax 816-363-0130).

Sample Preparation, NIR spectroscopy, and rapid determination of fat content are topics covered in a series of informational bulletins from Buchi Analytical, Inc. Rapid determination of fat content and individual fatty acids by the Caviezel method is covered in Bulletin 01-2000. The method is based on the fat definition given by FDA and involves extracting fat fromB the sample matrix using n-butanol as a solvent and simultaneously saponifying the fat in the presence of potassium hydroxide. The limiting step in the analytical pathway, particularly in food analysis, is sample preparation. Bulletin 05-2001 discusses the relevance of the mixing, grinding, and homogenization processes and their influence on the accuracy of the sample analysis. Bulletin 10-2001 discusses the importance of the right sample preparation of food samples for NIR spectroscopy. It evaluates the ideal sample preparation tool to find the optimal measurement conditions for NIR analysis of meat samples. For a copy of any of the bulletins, contact Buchi Analytical, Inc., 19 Lukens Dr., New Castle, DE 19720 (phone 302-652-3000; fax 302-652-87770).

Sterile-Pack Swabs for industrial surface sampling are available combined with a tube filled with rinse solution. Designed for hard-to-reach places, the swabs can fit easily into equipment recesses, nooks, and crevices. They are double wrapped, gamma-irradiated, and validated after exposure to the vaporized hydrogen peroxide atmosphere used during isolator facility decontamination cycles. The swabs are said to be ATP-free and have a shelf life of up to one year. For more information, contact BD Diagnostic Systems, 7 Loveton Cl., Sparks, MD 21152 (phone 410-316-4467).

Lab Blender, the Seward/Brinkmann StomacherB., is designed to blend and homogenize laboratory samples contained within sterile disposable bags. Additional applications include the removal of viruses and bacteria from tissues and organs; sterility testing of creams, ointments, or dry powder; and removal of organisms from swabs, filters, tissue biopsies, and fecal samples. Numerous versions are available to meet specific lab requirements and to accommodate different sample sizes. For more information, contact Brinkmann Instruments, Inc., P.O. Box 1019, Westbury, NY 11590-0207 (phone 516-334-7500; fax 516-334-7506).

Powder Rheometer, the FT3, provides means of assessing the flowability and processability of powders. It can evaluate flow properties in a small sample of material (10300 mL) by first pre-conditioning it to produce a consistent bed, thenB measuring the torque, force, and energy in the test chamber as a blade traces a precise helical path through the powder. Standard programs are automated, and the resulting data provide flow rate and compaction indices. For more information, contact Freeman Technology, Boulters Farm Centre, Castlemorton Common, Malvern, Worcestershire, UK, WR13 6LE (phone +44 (0) 1684 310860; fax +44 (0) 1684 310236; www.freemantech.co.uk)

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pH Meter, the IQ400, can be used as a handheld or benchtop pH meter. The system is a pH module coupled with a personal digital assistant. The meter accepts both traditional glass electrodes and rugged, non-glass, silicon-chip-sensor pH probes. Features include touch-screen graphics display, pop-up windows and on-screen troubleshooting guides for each function. Other features include high/low pH level alerts, recalibration alarms, automatic buffer recognition, save/recall of up to 9,999 records, and automatic temperature compensation. Data can be downloaded to a PC. For more information, contact IQ Scientific Instruments, Inc., 11021 Via Frontera, Suite 200, San Diego, CA 92127 (phone 858-673-1851; fax 858-673-1853 www.phmeters.com).

Moisture Analyzer, the Sartorius MA45, is designed for laboratory quality control applications covering incoming inspection and in-process control. It has a maximum weighing capacity of 45 g with 1-mg resolution, a temperature range of 40b�0;13;230B0C, and an infrared heat source for uniform heating of samples. It also has a choice of three modes—fully automatic, semiautomatic, and timer—for end-point determination of a measurement. For more information, contact Sartorius, Weender Landstrasse 94-108, D-37075 Goettingen, Germany (phone 49 551 308 0; fax 49 551 308 3289).

Liquid Chromatograph/Mass Spectrometer (LC/MS), Model 1200L, may be used to meet a variety of specific analytical requirements for life sciences and toxicology. The new system is available as a triple-quadrupole MS or a single-quadrupole MS that can be upgraded. Both configurations allow the user to switch from LC/MS to GC/MS and back with an interface change. Mass spectrometry is highly selective and sensitive. The LC/MS technique is of growing importance to life science researchers who value its ability to identify the large, polar, and nonvolatile molecules common to biological samples. The complementary GC/MS technique is used for identifying smaller, volatile, and semi-volatile compounds. The dual off-axis atmospheric pressure ionization interface is said to provide optimum droplet formation and maximum ion transmission. For more information, contact Varian, Inc., 2700 Mitchell Dr., Walnut Creek, CA 94598 (650-424-5286; www.varianinc.com).

Online Beverage Monitor, the new LAN 9000, is designed to measure the content of sugar (Brix) and CO2 in soft drink bottling operations. The monitor is said to increase productivity and profitability in beverage operations. Maintaining high quality standards for the production of diet beverages is said to be easier with the use of the instrument. The amount of each identified component is determined by the adsorption of the unique spectra of each component, which is proportional to its concentration in the beverage. The unit may be connected to a laboratory PC network for quality control analysis or networked to other internal or external locations for remote instrument control, data analysis, or diagnostics. For more information, contact OI Analytical, P.O. Box 9010, College Station, TX 77842-9010 (phone 979-690-1711; fax 979-690-0440).

Rheometer, the YR-1, may be used to measure yield behavior in foods. The instrument is designed to be used on the production floor by technicians. The user selects a test, lowers the vane spindle into the product, presses "Go", and the instrument displays the result in a single shear-stress number for the material's yield value. The instrument is said to have excellent repeatability and comes with software that allows the user to set up to 10 different test programs. For more information, contact Brookfield Engineering, 11 Commerce Blvd., Middleboro, MA 02346 (phone 508-946-6200 or 800-628-8139; fax 508-946-6262).

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