Development of a novel determination method for dioxin concentration

- enables more accurate and rapid dioxin determination than using the JIS method -

Key Points


The Institute for Environmental Management Technology [Director: Takashi Ibusuki] and Human Stress Signal Research Center [Director: Etsuo Niki] of the National Institute of Advanced Industrial Science & Technology (AIST) [President: Hiroyuki Yoshikawa]succeeded in rapid and accurate determination of dioxin concentration in actual environmental samples (fly ash from waste incinerators extracted and cleaned up with a accelerated solvent extractor) using QCM sensors. This development enables highly accurate and rapid dioxin determination in proportion to the GC/MS method in the JIS standard. It is hoped that it will make on-site determination of dioxins possible.

Acknowledgements: this development benefited from assistants of Hitachi Kyowa Engineering Co., Ltd. [President: Katsutoshi Kataoka] for GC/MS analysis of dioxins in environmental samples, Daiichi Fine Chemical Co., Ltd. [President: Yuichiro Takeda] for ELISA detection of environmental samples, and Nihon Dempa Kogyo Co., Ltd. [President: Toshiaki Takeuchi] for preparation of QCMs for dioxin sensors.

So far, dioxin concentration and toxic equivalent quantity in the environment could only be measured by the GC/MS method (JIS method).

Analysis results of dioxin concentration in environmental samples by the QCM method showed good correlation with the standard GC/MS method and ELISA, which are conventional dioxin determination techniques. The amount of waste to be incinerated was smaller because of the minute quantity of samples (not more than 10μL). Furthermore, analysis was completed in six hours (in contrast to about one week by the standard JIS method) from sample collection.

A simple measuring device for dioxins in environmental samples with the QCM sensor (dioxin sensor) is developed.

The dioxin sensor comprises a main unit of a low-cost battery-powered sensor (see the attached photo) and a single-round sensor chip for dioxins. It is expected that it will make on-site determination possible in compliance with the Law Concerning Special Measures against Dioxins which will be strictly administered from this December.

The result will be presented in QCM2002, which will be held on July 24-25, 2002, in Brighton, England.

Future, research is planned for: optimal determination equipment for high- and low-concentration dioxin analysis; applying that determination method to “High-Concentration Dioxin Analysis in Fly Ash from Waste Incinerators and in Soil around Such Facilities” and to “Low-Concentration Dioxin Analysis in Water, Air, Underwater Sediments, and Tissue-Derived Samples”; and comparison of data acquired by GC/MS, ELISA, and quartz crystal methods. Moreover, there is a plan to develop sensors for disease marker proteins which appear in blood along with diseases. This is accomplished to meet the needs of an ever-aging society by altering the antibody to be immobilised on the QCM sensor-chip for the purpose of home health care.

—:See attached Glossary

Summary Figure
Figure: Appearance of the Dioxin Sensor Developed by AIST


The conventional analysis method for dioxins in compliance with the JIS method requires copious quantities of work, time and cost because environmental samples need to be extracted from target objects and cleaned before measurement by the GC/MS method. Also, in order to calculate dioxin concentration and its toxic equivalent quantity, it is necessary to determine concentration of each dioxin isomer and multiply it by its toxicity coefficient to convert it into a value in 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). As dioxins are very toxic, the analysis procedure requires experienced analysts with high skill to perform analyses in a laboratory under safety controls. In Japan, there are few organizations that analyse samples on a contract basis. It costs approximately ¥250,000 per sample for an analysis which takes about three months. Therefore, it has been difficult to obtain sample analysis for common citizens, consumer cooperatives, agricultural cooperatives, industrial waste disposal contractors, and those construction sites and waterworks-related companies which require soil analysis.

On the other hand, enzyme-linked immunosorbent assay (ELISA) is widely used as a simple determination method of dioxins. This method utilises highly specific immunoreaction that binds with an antigen; the antigen is recognised as foreign when foreign substances such as chemicals enter the body of an organism. Immunoreaction is also used in blood analysis for medical diagnosis and in BSE investigation, but it is supposed that pretreatment (removal or extraction of other components) presents difficulties with some samples.

Anti-2,3,7,8-TCDD monoclonal antibody, which is the only one available in the market, shows high reactivity with 2,3,7,8-TCDD, the most toxic dioxin, but its reactivity with other dioxin isomers and homologues is low. For that reason, it was difficult to evaluate the toxic equivalent quantity; simple, sensitive determination of dioxins seemed difficult. This study made positive use of cross-reactivity of the anti-dioxin antibody and proved it could be utilised not only for determination of dioxin concentration in environmental samples, but also for estimation of the toxic equivalent quantity.

AIST (formerly, the National Institute of Materials and Chemical Research) investigated the feasibility of ultrasensitive sensors for dioxin measurements and demonstrated their usefulness under the project “Preliminary Investigation on Utilization of Sensors in Environmental Determination of Dioxins” in preliminary investigation of the Environment Agency in the second half of FY1999. Moreover, AIST developed a method for highly sensitive and simple determination of dioxins using QCM sensors in the course of performing the succeeding project “Study on Measures against Environmental Risk Using the Sensing System of Dioxins and Endocrine Disrupting Chemicals” under a grant for scientific research on global environment protection from the Ministry of the Environment (FY2001-2005). It examined optimal conditions through demonstration experiments for sensor usage using samples that contained high- and low-concentration dioxins which were extracted from actual environmental samples and pretreated.

Contents of the Project

Conditions for measurement of 2,3,7,8-TCDD were examined using a QCM on which anti-2,3,7,8-TCDD monoclonal antibody and its stabiliser were immobilised; it was shown that measurement of 2,3,7,8-TCDD concentration was possible in a concentration range from 0.1ng/mL to 100ng/mL. Such concentration was sufficient for the sensitivity required for environmental monitoring at around 80ng-TEQ, which was the environmental standard for soil. Using this method, determination conditions for the QCM were examined with samples that contained high-concentration dioxins prepared from actual environmental samples (fly ash from waste incinerators) through pretreatment and cleanup by an accelerated solvent extractor. The analysis result of dioxin concentration in environmental samples by the QCM method showed good correlation with dioxin concentration determined by the conventional GC/MS method and with the current simple determination method ELISA. The amount of waste to be incinerated was smaller because of the minute amount of samples (not more than 10μL). Furthermore, analysis was completed in six hours (in contrast to about one week by the conventional JIS method) from sample collection.

Future Projects

The current method with anti-dioxin antibody utilises antigenic specificity of antibody in immunoreaction, but the antibody has cross-reactivity to compounds that have similar chemical structure with the antigen. Therefore, the differential composition of dioxins in the target object may cause errors in results such as excessive or insufficient response. In order to establish a reliable estimation range for determination of dioxin concentration and toxic equivalent quantity in environmental samples by the QCM method, comparative study with analysis data of actual environmental samples by the GC/MS method will be continued along with refinement of the equipment. Measurement of dioxin concentrations in sedimentary soil, air, water, fish meat, breast milk, and blood require higher sensitivity to enable measurement in the picogram range, 10-100 times more sensitive than the current detection range in the nanogram (10-9g) range. Development is anticipated for equipment for dioxin determination that has a femtogram (10-15g) to picogram (10-12g) measuring range of dioxins so that it can measure such samples. Develop is also anticipated for a technology for miniaturization of equipment for further reduction of measuring time and sample amounts. At the same time, there is a project to prepare anti-dioxin antibody other than the available ones for tetrachlorinated dioxin. They would enable recognition of penta- or hexachlorinated dioxins which are commonly found in fly ash from waste incinerators and have high toxicity coefficients. Other efforts to evaluate performance of sensor devices with the antibody will be boosted for further development to improve immunoreaction selectivity.


Dioxin concentration and toxic equivalent quantit
Dioxin is a generic name for polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran and co-planar polychlorinated biphenyl (co-planar PCB). There are many species of dioxins that differ by chlorinated sites or their numbers, and their toxicity differ from one another. Coefficients that convert toxicity of dioxins by the toxicity of the most toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) referred to as 1 are used for evaluation of toxicity of dioxins as a whole. Data of amounts and concentrations of various dioxins are expressed as a toxic equivalent quantity (TEQ) that is the sum of the toxicity of dioxins using toxicity coefficients. Dioxins have not been produced intentionally up to now, but they are formed along with manufacture or combustion of other chemicals. Presently, there are guidelines for waste incinerators on the structure of their facilities and equipment and on incineration conditions to prevent dioxin generation. Also, paper or pulp plants are given guidance to minimise use of chlorine, which causes generation of dioxins.[back]
GC/MS method (JIS method)
Japanese Industrial Standards (JIS) are provided as the determination method for dioxins and coplanar PCBs in JIS K0311 for exhaust gases, and in JIS K0312 for water for industrial use and industrial effluents. Both are definitions of measurement methods for dioxins by gas chromatography/mass spectrometry (GC/MS). The GC/MS used here is defined as a double-focusing mass spectrometer that uses a capillary column as the GC column and has resolution of not less than 10,000x. There are many forms of dioxin-containing samples, such as air, soil, water, underwater sediment, fish, meat, blood and breast milk; they cannot be directly introduced into GC/MS. Thus, it is necessary to extract, pretreat, and clean-up dioxins from samples.[back]
Anti-dioxin antibody
Dioxins are harmful to organisms. An antibody will be produced when enter a living body. This is called anti-dioxin antibody. In this study, anti-2,3,7,8-TCDD monoclonal antibody was immobilised on the sensor chip for dioxin determination. The antibody combined selectively with 2,3,7,8-TCDD that was the most toxic; it also accounts for the major part of environmental samples of dioxins.[back]
Quartz crystal microbalance (QCM)
Quartz crystals are used as stable oscillation devices for a frequency standard in various electronic circuits such as clocks, computers, telecommunications equipment, and measuring instruments. They are known as ultramicrobalances because they convert the weight adhered on their electrodes into a quantitative change in oscillation frequency; they are used in thickness-meters and chemical sensors. Chemical sensors usually utilise an AT-cut quartz crystal of 9 MHz fundamental frequency which is easy to handle; thereby, a 1 ng adhesion of material on the crystal can be directly detected as a 1 Hz decrease in oscillation frequency. A quartz crystal with a larger fundamental frequency or overtone oscillation is used to prepare more sensitive sensors.[back]
10μL is equal to one thousandth volume of 1mL. Minute quantities are expressed as follows: 1 ng is one billionth of 1g, 1pg is one trillionth of 1g, 1 fg is one quadrillionth of 1g, respectively.[back]
On-site determination
Recently, the demand and need to determine quantities and obtain results right after collecting samples are growing in the work front of analysis. In laboratory analysis, collected soil or water samples are taken back to the laboratory to be analysed for concentration measurement certification by the JIS method for comparison with environmental criteria. On the other hand, on-site analysis allows quick determination of the situation; it consequently facilitates efficient investigation flow.[back]
Law Concerning Special Measures against Dioxins
In order to reduce dioxins, the Japanese government promulgated the “Law Concerning Special Measures against Dioxins” on July 16, 1999; it established criteria as the basis of measures against dioxins as well as necessary regulations and measures against contaminated soil for prevention and remediation of environmental pollution by dioxins.[back]
Accelerated solvent extractor
Dioxin determination by immunoassay requires extraction, cleanup and measuring procedure, as is the case with analysis by the GC/MS method. It is important for analysis with high sensitivity that inhibitors extracted along with dioxins affect immunoassay. Time for each procedure as well as immunoassay needs to be cut down to speed up dioxin analysis. In the accelerated solvent extractor method, dioxins are extracted from solid samples by solvents at high temperature under high pressure in a short period of time.[back]
Enzyme-linked immunosorbent assay (ELISA) is a simple measuring method that utilises antigen-antibody reaction. In this process, antibody of the measuring object as antigen is immobilised on solid phase base material and a secondary antibody labeled with fluorescent pigments, radioactive materials, enzymes, etc. is used. Many reaction procedures and reagents are required because labeled compounds are used. Analysis kits for dioxins are now available from several suppliers. In Japan, the GC/MS method is officially authorised and ELISA is used as supplement of the GC/MS method.[back]
This conference will be held in Brighton, England. Latest studies will be brought together on non-labeled real time analysis with Quartz Crystal Microbalance (QCM), especially with a focus on new analysis methods relevant to pharmacology and biochemistry, academic studies, and industrial applications. Twenty oral presentations and sixteen poster presentations are planned. It is supported by the Royal Society of Chemistry, the Royal Pharmaceutical Society, the University of Brighton, and GlaxoSmithKline.[back]
Antibody selectively combines only with its antigen, but it also combines in error with compounds that resemble the antigen in chemical structure. The ratio of combination in error is expressed as the cross-reactivity of the compound to the antigen.[back]

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