Abstract: With the increasing emphasis on China's water resources environment and drinking water quality safety, water biological toxicity analysis has gradually become one of the methods for evaluating water quality pollution, and it has also been more and more widely used in water quality monitoring of emergency water treatment. This article mainly summarizes the latest biological toxicity detection technology at home and abroad, and introduces the world's latest chemiluminescence toxicity analysis method. At the same time, it carries out the technical theory of bacterial toxicity and chemiluminescence two toxicity analysis methods, as well as water pollution and water supply. Specific analysis of the application in emergency response.
Keywords: Toxicity Monitoring, Bacterial Luminescence, Chemiluminescence, Portable Toxicity Analyzer
1 Necessity of biological toxicity monitoring
With the development of modern industry, the use of chemical substances is increasing, and the aquatic ecosystem on which humans depend is increasingly polluted, and sudden environmental pollution accidents occur from time to time, such as human poisoning and natural disasters. Sudden changes in water quality, especially the pollution of environmental water bodies caused by accidents in the production, storage and transportation of petrochemical raw materials, finished products and toxic and hazardous products. This requires us to quickly respond to all kinds of sudden environmental pollution accidents and minimize various economic losses or social impacts. For decades, the sensitivity of various physical and chemical analysis methods has become higher and higher. Most researchers are concerned about the toxic effects of single pollutants on organisms and ecosystems. However, organisms in the environment are often exposed to multi-component pollution In a mixed system in which things coexist, rather than a simple single system. The toxic effect produced by the mixture system is a comprehensive result of the antagonistic, additive, synergistic or inhibitory effects of all component pollutants. Even if the single component in the mixture system is at a concentration without toxic effect, the total toxic effect of the component on the mixture still has Certain contribution [1-4]. Therefore, it is very urgent and necessary to develop new effective methods to quickly and accurately evaluate the toxicity of various pollutants.
This article mainly studies and discusses the latest biological toxicity monitoring technology at home and abroad.
2 Biological toxicity monitoring technology
For the measurement and evaluation of biological toxicity of toxic substances in the environment, aquatic organisms such as plankton, algae and fish are generally used to evaluate the toxicity of environmental pollutants based on their morphology, motility, changes in physiological metabolism or mortality as indicators. These methods once became one of the necessary means to evaluate environmental pollution, but these methods are relatively cumbersome, the detection time is long, the detection cost is high, and the results are unstable, and the repeatability is poor, making it difficult to popularize and apply, and not suitable for conventional Inspection, especially on-site emergency monitoring [5]. In response to the shortcomings of traditional biological toxicity detection methods and the needs of on-site emergency monitoring, some fast, simple and economical modern detection methods have been gradually developed, such as luminescent bacteria toxicity detection methods, chemiluminescence toxicity detection methods, etc. Among them, luminescent bacteria have attracted much attention because of their unique physiological characteristics and the characteristics of perfect matching with modern photoelectric detection methods. The chemiluminescence toxicity monitoring method is the latest toxicity evaluation technology, which makes up for some of the limitations of the bacterial luminescence method in the field, and can pollute the water source and drinking water caused by sudden events or man-made damage within the first time. The assessment of the incident has attracted more and more attention from various environmental fields.
2.1 Bacterial luminescence detection technology
Luminous bacteria comprehensive toxicity detection technology is a toxicity detection technology based on the bacterial luminescence biological sensing method, which can effectively detect sudden or destructive environmental pollution. The luminescence process of luminescent bacteria is a physiological process of metabolism in the bacteria. It is the process of photorespiration. It is a side branch on the respiratory chain. The wavelength of this light is around 490nm. This luminescence process is extremely susceptible to the influence of external conditions. Anything that interferes with or damages the bacterial respiration or physiological process can cause the luminescence intensity of the bacteria to change. When toxic and harmful substances come into contact with luminescent bacteria, the toxic substances in the water sample will affect the metabolism of the luminescent bacteria, and the decrease in luminous intensity is proportional to the concentration of the toxic substances in the sample. The reaction mechanism is shown in the following chemical equation:
FMNH2 + O2 + R-CHO → FMN + R-COOH + H2O + Light
In a nutshell, the bacterial bioluminescence reaction is catalyzed by molecular oxygen, catalyzed by luciferase in the cell, and oxidizes the reduced flavin mononucleotide (FMNH2) and long-chain fatty aldehyde to FMN and long-chain fatty acid, while releasing maximum luminescence. Blue-green light with an intensity of around 490nm [6].
At present, the luminescent bacteria method has become a simple and rapid biological toxicity detection method, widely used in quality inspection, environmental monitoring, aquaculture and other fields, and is included in the international standard ISO11348, China ’s national standard GB / T15441- 1995, the German national standard DIN38412. In China, it is currently based on international ISO standards and China's national standards, especially international ISO standards.
The detection principle of the ISO11348 standard is that, at a temperature of 15 ° C, a non-toxic reference solution is used for comparison. After the sample or its diluted solution is contacted with Vibrio Fischeri (Vibrio fischeri) for 15 minutes or 30 minutes or 5 minutes, the actual sample is measured for luminescence. Bacterial inhibition rate. The toxicity level of water quality is expressed as LID value (dilution factor of the sample when the inhibition rate is reduced to 20%), EC20 or EC50 value (concentration of the sample when the inhibition rate is 20% or 50%). The higher the LID value, the lower the EC value, indicating that the sample is more toxic. The national standard GB / T15441-1995 adopts the bacterium Brevibacterium luminescens T3, and the toxicity level of water quality is characterized by the equivalent mercury chloride concentration or EC50 value.
2.2 Characteristics of Bacterial Luminescence Biotoxicity Instrument
At present, most of the common biological toxicity analyzers at home and abroad are based on the principle of bacterial luminescence. Generally, there are desktop toxicity instruments suitable for laboratory testing and portable toxicity instruments suitable for field use. The tabletop toxicity analyzer is mainly used in the laboratory. In a 15 ℃ thermostat, the toxicity analysis of the sample is performed according to the ISO standard process, and the toxicity level LID value or EC value of the sample is calculated. The portable toxicity meter is mainly used for routine rapid monitoring on site or emergency monitoring of emergencies. It uses simplified ISO standard process for analysis. Generally, it is not equipped with a thermostat to detect the luminescence inhibition rate of water samples to luminescent bacteria.
For toxicometers using the luminescent bacteria method, their main characteristics are:
(1) Strains: Different strains are used, and the effects of poisons in water on their luminescence process are different. Therefore, in order to make the data more authoritative and comparable, the strains that meet the standards are generally used. At present, the three kinds of luminescent bacteria commonly used in China are: Luminescent luminescent bacteria, Vibrio fischeri and Vibrio qinghai. Among them, Bright Photobacterium is used in the GB / T15441-1995 standard; Vibrio fischeri is used in the international ISO11348 standard; Vibrio qinghai belongs to freshwater bacteria, which is a kind of luminescence extracted from fish in Qinghai Lake in China Species, currently this kind of bacteria is generally used in drinking water in China. Domestic bioluminescence photometers generally use the light-emitting bacterium T3 race that meets the national standard, and some also use Vibrio qinghai. The toxicity instruments abroad are basically Vibrio fischeri that conforms to the ISO standard, and some of them use Photobacterium brachii. The sensitivity of the bacteria is relatively high, but the recovery time is up to 3 hours, which is not conducive to the application of rapid detection on site.
(2) Anti-interference ability: Toxicity measurement is to measure the relative luminosity of the sample through a photometer, so the color or turbidity of the water sample will cause great interference to the test results, which is also the actual water sample measurement of all toxic meters The biggest interference encountered. At present, only a desktop biotoxicity meter manufactured in Germany has the function of compensating for chromaticity and turbidity.
(3) Certification: For on-site characterization of water quality, USEPA discourages the use of on-site testing techniques whose properties (such as false positives or false negatives) have not been verified.
(4) The expression form of the results: because it is mainly for rapid screening on the spot, the portable toxicity analyzer basically uses the simplified ISO11348 standard process to quickly assess the toxicity of the sample, and generally determines the inhibition of the concentration of a sample. Rate, the so-called single-point result expression, the higher the inhibition rate, the more toxic the sample solution. All portable toxicity meters basically use this method. In addition to the single-point expression method, a toxicometer from a US brand has also developed a unique three-point expression method similar to the ISO standard, and uses a unique color card (see Figures 1 and 2), Three points fall in different color areas to determine the toxicity and danger of the water sample. This method is more intuitive and makes it easier for users to determine the toxic strength and treatment of water samples.
(5) Detection speed: On-site applications generally require the faster the detection speed, the better. Since the bacterial luminescence method is used, the operation process is basically the same. It is only the preparation process of the bacteria that affects the detection speed. In general, the bacterial species need to undergo a recovery and dilution process. Among all the bacterial species, only the recovery time of Photobacterium brachyphilus requires up to 3 hours, which is not conducive to rapid on-site detection.
(6) The portability of the instrument: In the field work, the compactness, portability, and ease of use of the instrument are very important. The portable instruments are basically small and compact, and they can all use batteries as a power source, weigh 2 to 3 kg, and are equipped with a portable case. The instrument and reagents are built in the portable case for easy portability. These functions will increase the convenience of on-site use.
(7) Auxiliary functions of the instrument. In addition to the bacterial luminescence method, some toxicity instruments also have some auxiliary functions to provide more information for quickly judging the toxicity of the water body. For example, some toxicity analyzers have ATP (adenosine triphosphate) analysis function, which can determine the number of active organisms in a unit sample, but this function is not used much in water quality toxicity analysis. And some of the supporting equipment of some toxicity meters are also equipped with a variety of accessories for on-site water quality analysis, such as pH meter, TDS meter, arsenic rapid test paper, residual chlorine & total chlorine colorimetric disk, colorimeter disk, pesticide / neurotoxic agent test paper, etc. , To provide favorable information for on-site comprehensive toxicity analysis and judgment.
2.3 The latest biological toxicity detection technology-chemiluminescence method
Although the on-site water quality comprehensive toxicity analysis technology of the bacterial luminescence method is widely used in environmental monitoring, the luminescent bacteria reagents required by this method need to be stored under the conditions of -18 ~ -20 ℃, and the recovery temperature must also be within 3 ~ It is carried out under the condition of 8 ℃, which is very harsh for the field environment, which affects the repeatability and stability of the experimental data. In recent years, there has been a new toxicity evaluation technology-chemiluminescence method, which is based on the intensity of the radiation generated by the chemical reaction to determine the analysis method of the corresponding substance content in the reaction. It has high sensitivity, simple equipment, and fast analysis speed. Features such as wide linear range.
This latest water quality biological toxicity analysis technology was originally developed for the military and widely adopted by the US and British military. Based on the successful application of this method in the military, the United States began to promote the use of the EPA, urban emergency centers, water companies, municipal water supply companies and other fields. Based on the advantages of chemiluminescence method, a certain American water quality analysis instrument company developed and successfully developed an on-site water quality biological toxicity test instrument using chemiluminescence method. In addition to the toxicity analysis function of the bacterial luminescence method, the instrument also has the latest toxicity analysis function of the chemiluminescence method! This technology has not only passed the USEPA Environmental Technology Certification Project (ETV), but also the portable water quality toxicity analyzer has become the United States Environmental Protection Agency. (USEPA) Water quality analysis equipment recommended in the emergency response draft.
The principle of chemiluminescence is based on the catalysis of horseradish peroxidase, the luminescent reagent and the oxide react chemically, and flash (chemiluminescence) occurs during the reaction. When there are toxic substances in the sample, it will affect the progress of the reaction, and then affect the luminous intensity. The toxic intensity of the sample can be determined by the change of the luminous intensity [7-8].
The analysis process of the chemiluminescence method is very simple, no special temperature conditions are needed, just add 1mL of water sample to the test tube, add 0.1mL of CT1, CT2 and CT3 reagents respectively, put the test tube into the instrument and scan for 4min For comparison with a non-toxic reference solution, the final test result will be expressed in terms of relative luminous intensity—inhibition rate, as shown in Figure 3.
Compared with bacterial luminescence, chemiluminescence has the following advantages:
(1) The test speed is fast, the first measurement result can be obtained within 5 minutes, while the traditional light-emitting bacteria method requires at least 15 minutes;
(2) The analysis can be carried out at normal temperature without the need of a thermostat;
(3) The reagents used can be stored at room temperature;
(4) The reagent is stable, the test is convenient, and the data repeatability is good, avoiding the bacterial luminescence method to obtain different data due to the use of different bacterial species.
3 Practical application of toxicity analyzer
The bacterial luminescence method has been applied to the toxicity detection of the water environment as early as the 1970s. Therefore, at present, the method is more widely used than the chemiluminescence method. However, the luminescent bacteria reagents used for the luminescence of bacteria need to be stored under the conditions of -18 ~ -20 ℃, and the recovery temperature must also be carried out under the conditions of 3 ~ 8 ℃, which is very good for the laboratory It is easy to implement, but it is very harsh for the on-site environment, which is not conducive to the use in the field. Therefore, this technology is more suitable for the laboratory. The chemiluminescence method is completely developed for the needs of field applications. Its reagents can be stored at room temperature and can be analyzed at room temperature, so it is more adaptable to various field environments.
At present, a brand has launched the latest two-in-one instrument, and one instrument has both the two technologies described above-bacterial luminescence and chemiluminescence. This will be more practical for customers with different requirements.
3.1 Water quality emergency monitoring
In emergency monitoring, it is the most critical to obtain the toxicity data of the polluted water in the first time. Therefore, the current application of the toxicity analyzer is mainly focused on the emergency monitoring on the spot. In the early stage of development, the field application mainly used the bacterial luminescence method, but the storage conditions of its reagents became more and more restrictive conditions for its field application. With the advent of the simple chemiluminescence method, it makes up for the shortcomings of the bacterial luminescence method, and has attracted more and more attention from various customers.
For example, in 2010 Shanghai World Expo, in order to ensure the safety of various water sources and drinking water during the Expo, the only tap water supply source in Puxi in the Expo area-Nanshi Waterworks, Shanghai Sanitary Supervision Institute, Shanghai Fengxian Hydrological Station, Shanghai Changning District Environmental Monitoring Station, etc. They have purchased instruments that have both toxicity analysis technologies, and are ready for emergency water safety and environmental pollution accidents.
In addition, in order to ensure the water supply safety of another international event in 2010, the Guangzhou Asian Games, the Guangzhou Water Quality Monitoring Center initially inspected various brands of toxicity analyzers and purchased a portable toxicity device with both technologies. Analyzer. Through an initial exploration of the water quality at the end of the water pipe network in Guangzhou, the feasibility of this method was tested, and the feasibility and convenience of the use of chemiluminescence in the field and long-term water quality background monitoring were confirmed. In order to ensure the smooth progress of the Guangzhou Asian Games, the center needs to monitor more than 100 pipe network end points throughout Guangzhou to ensure the safety of water supply during the Asian Games. In order to accomplish such a difficult task, the center continued to purchase 8 portable toxicity analyzers with chemiluminescence method, which provided a very comprehensive guarantee for the safety of Guangzhou's water supply.
3.2 Long-term background monitoring application of water quality
For the comprehensive toxicity analyzer, in addition to on-site emergency monitoring applications, there is also a very important application area-long-term background monitoring of water bodies. The current pollution range of water quality is showing an increasing trend, making the pollution of various water bodies more and more serious, especially drinking water source water. Therefore, the establishment of background monitoring files for water quality changes from morning to night, every day to every season, and even every year for various water bodies will provide more information for environmental monitoring and other fields. For example, you can track pollution incidents and trace possible sources of pollution; evaluate industrial sites; quickly respond to water quality impacts caused by various natural disasters; you can also evaluate existing and potential source water and determine the most suitable water supply system Processing technology and so on.
Since such applications will consume a lot of reagents, human resources, time, etc., it can be the biggest challenge for such applications to complete the water quality monitoring of each stage in the shortest time under the minimum reagent cost and labor cost. . At this time, the advantages of chemiluminescence will become more and more prominent.
With the end of the World Expo and the Asian Games, the above-mentioned toxicity analyzer will gradually be put into the application field of long-term background monitoring of water quality, so that the application of the above instruments can be maximized, and it is also used in Shanghai and Guangzhou. Environmental monitoring makes a greater contribution.
4 Application prospects of biological toxicity analyzer
Compared with other traditional biological monitoring methods, the luminescent bacteria method has the characteristics of quickness, simplicity, and sensitivity, which makes it widely used in water quality and environmental assessment. With the development of technology, more advanced chemiluminescence analysis methods will be gradually popularized and applied in on-site emergency monitoring and long-term background monitoring of water quality comprehensive toxicity analysis due to its faster, simpler and more sensitive characteristics. In short, the combination of chemiluminescence and bacterial luminescence will provide a more comprehensive, rapid, and accurate toxicity analysis for environmental monitoring. It is believed that these two test methods will play a greater role in environmental monitoring.
references:
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[2] Altenburger R, Backhaus T, Boedeker W. Predictability of the toxicity of multiple chemical mixtures to Vibrio fischeri: mixtures composed of similarly acting Chemicals. Environ Toxicol Chem, 2000, 19: 2341-2347.
[3] Faust M, Altenburger R, Backhaus T. Predicting the joint algal toxicity of multi-component s-triazine mixtures at low-effect con-centrations of individual toxicants. Aquat Toxicol, 2001, 21: 13-32.
[4] Wang Bin, Yu Gang, et al. Quantitative structural activity correlation and joint toxicity prediction of alkyl alcohol compounds, Science Bulletin, 2006, 51 (13): 1513-1518.
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[8] Li Yixin, Fang Yunzhong, Experimental observation of chemiluminescence in photosensitization reaction of hematoporphyrin derivatives, Bulletin of Academy of Military Medical Sciences, 1987, 11: 446.
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