CN117487793A - Combined acute toxicity test method for field water quality on-site detection - Google Patents
Combined acute toxicity test method for field water quality on-site detection Download PDFInfo
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- CN117487793A CN117487793A CN202311424048.0A CN202311424048A CN117487793A CN 117487793 A CN117487793 A CN 117487793A CN 202311424048 A CN202311424048 A CN 202311424048A CN 117487793 A CN117487793 A CN 117487793A
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/084—Polymers containing vinyl alcohol units
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention belongs to the technical field of water quality detection, and discloses a combined acute toxicity test method for field water quality on-site detection, which comprises the following steps: s1: preparing a portable handheld water quality acute toxicity detector; s2: preparing a plurality of luminous fungus gel beads in batches in advance; s3: preparing a water sample to be detected; s4: naturally thawing frozen luminous fungus gel beads in batches; s5: placing the detection pore plate, the water sample to be detected and the luminous fungus gel beads into a detection bin; s6: and respectively collecting and recording fluorescence change data of the luminous strains in each detection hole, and calculating to obtain concentration data and combined toxicity effect corresponding to toxic substances in the target objects to be detected in each detection hole. According to the invention, through the cooperative improvement of the detector and the luminous fungus gel beads, detection equipment, detection materials and detection operation steps are greatly simplified, the adaptability to environmental conditions and detection efficiency are improved, and the batch and rapid combined detection of complex water samples under the field condition of water quality toxicity can be realized.
Description
Technical Field
The invention belongs to the technical field of water quality detection, and particularly relates to a combined acute toxicity test method for field water quality on-site detection.
Background
Since the 60 s of the 20 th century, environmental problems are increasingly emphasized, and rapid, sensitive and low-cost biological detection methods are urgently needed, so that the research of luminescent bacteria for detecting environmental pollution toxicity is more and more, and several marine luminescent bacteria such as the end of the 70 s, the luminous luminescent bacillus, the vibrio freudenreichii and the like are used for detecting the water environment toxicity. With the intensive research and application of the luminescent bacteria detection method, the application range of luminescent bacteria detection of toxic substances is continuously expanded, and luminescent bacteria are also adopted as an important means for rapidly detecting toxicity in the fields of soil, air, solid waste pollutants, food safety, drinking water safety and the like.
The currently commonly used luminescent bacteria toxicity test is a new method for monitoring environmental pollution and detecting pollutant toxicity by microorganisms which are rising after the 70 th century of 20 th century. In 1978, beckman company in united states of america introduced a fully functional bioluminescence photometer "Microtox", and from this point on, this acute toxicity test technique was rapidly popularized worldwide. Therefore, the light-emitting bacterial toxicity test is also called a Microtox test. The phototoxicity test of the luminous bacteria by adopting a modern photoelectric detection means (bioluminescence photometer) is one of the bioassay methods in toxicology. The method is quick, simple, sensitive and cheap, has important significance in the aspects of screening toxic substances, evaluating the biology of environmental pollution and the like, and is concerned by researchers in various countries. In month 3 of 1995, the national environmental protection agency and the national technical supervision agency set the toxicity test of luminescent bacteria as a standard method for water quality monitoring (GB/T15441-1995).
The prior art luminescent bacterial method is used for acute toxicity test methods, including conventional laboratory acute toxicity tests and comprehensive biological toxicity on-line monitoring automatic analysis instruments related to the luminescent bacterial method. The national standard "method for measuring acute toxicity of light-emitting bacteria" of water quality (GB/T15441-1995) prescribes a method for measuring acute toxicity of light-emitting bacteria applicable to measuring acute toxicity of water environment such as industrial wastewater, wastewater containing body and the like and soluble chemical substances under experimental conditions, but the standard method is only applicable to laboratory acute toxicity analysis test of water samples and chemical substances, for example, the document of China patent application publication No. CN 111925924A discloses a comprehensive water quality biological toxicity test device and a test method based on light-emitting bacteria, the provided test device comprises a device main body, a first activation device, a second activation device, an activated bacteria liquid storage tank, a to-be-tested sample preparation device, a water quality sampling device, an analysis test device, an interaction device and a cleaning device, wherein the first activation device comprises a light-emitting bacteria storage tank, an activation chamber I, a nutrient solution tank and a buffer tank, the second activation chamber I and a heating device, and the activation bacteria liquid storage tank I are respectively connected with the activation chamber I, the to-be-tested sample preparation device comprises a preparation chamber, a driving motor, a to-be-tested sample tray, a to-be-tested sample container, a water quality test tube, a liquid adding tube, an activation liquid adding tube and a water tube. Therefore, the testing device and the testing method in the prior art are complicated, and are difficult to be suitable for rapid toxicity analysis and testing in the field. In addition, the prior art adopts the preparation of the working bacterial liquid of the luminescent bacterial lyophilized powder, which requires longer time, and has corresponding requirements on conditions such as re-dissolved ambient temperature, air cleanliness and the like, so as to ensure that a sufficient number of active bacterial strains are obtained and are not polluted by miscellaneous bacteria in the environment. For example, the publication CN102465167 a discloses a rapid, high-throughput acute toxicity test method for luminescent bacteria, which adopts luminescent bacteria lyophilized powder or cryopreserved cryopreservation tube, rapidly thaws at 20 ℃, adds 1.0mL of resuscitation solution into the tube, after mixing uniformly, carefully transfers the mixed solution in the tube into 100mL of resuscitation solution, and stirs at 500 rpm for 30 minutes at 20 ℃; then, on a 96-micro-pore plate, transferring 245 mu L of sample solution to be tested into the micro-pore plate by using an 8-channel pipettor, then transferring 5 mu L of working bacterial liquid into each hole rapidly, and starting timing; the luminescence intensity of each well of the microplate was measured on a chemiluminescent microplate reader after 15 minutes of exposure and the results were recorded. Therefore, the preparation time of the working bacterial liquid is long (more than 30 minutes), the required exposure time is long (more than 15 minutes), and the traditional chemiluminescent enzyme-labeled instrument adopted by the method cannot be carried to field work.
In the prior art, the commonly used luminescent bacteria are marine bacteria, the marine luminescent bacteria are utilized to carry out biological toxicity test on environmental samples, a certain amount of NaCl (2% -3%) is required to be added in the detection process to maintain the activity of the marine luminescent bacteria, the detection of the toxicity of pollutants in fresh water system samples is influenced and limited, and the defect of the marine luminescent bacteria application method can be avoided just by the freshwater vibrio vulnificus found in China, so that the advantage that the marine luminescent bacteria can emit light in the fresh water is exerted. Vibrio qinghaiensis can normally emit light in a fresh water system, and the temperature application range is wide, and the vibrio qinghaiensis can emit light at 10-35 ℃; as a freshwater type luminous bacterium, vibrio qinghaiensis is effective for Na + In addition to the characteristic, the fresh cultured vibrio qinghaiensis can emit light well in distilled water and the stable light emission can be maintained for more than 30min, so that distilled water can be used as a blank control in the detection of a fresh water sample, and the fresh water sample can be tested by directly adding the vibrio qinghaiensis without any additional additive. In addition, the strain has wider adaptability to the pH value of the environment, and can normally emit light in the pH value range of 6.0-9.0, namely the pH value has negligible influence on the light emission of the vibrio qinghaiensis, so that the strain can be used without adjusting the pH value of a sample And detecting. The vibrio qinghaiensis is the only non-pathogenic freshwater luminous bacteria, the freeze-dried powder is safe and reliable in the transportation and use processes, and the waste bacterial liquid does not need special treatment and cannot cause secondary pollution.
In the prior art, many research methods can solve the problem of acute toxicity detection of single toxic substances (inorganic substances or organic substances), but for combined detection of two or more toxic substances simultaneously, and determining the combined toxic effects of the two on the whole, effective research means are not available. And the current luminous bacteria liquid detection method is adopted, so that corresponding results can be obtained only by carrying out detection for multiple times, and more monitoring materials and detection time are required to be consumed.
Vibrio qinghaiensis Q67%Vibrio qinghaiensis sp.nov. Q67) The characteristic that the luminous intensity of the freshwater luminous bacteria changes along with the concentration of the poison is determined in China, so that the freshwater luminous bacteria can be used as an indicator strain for water quality detection, blue-green light can be continuously and stably emitted when the strain is in a normal environment, and the maximum emission wavelength is 485nm; however, once toxic and harmful substances are encountered, the light-emitting process is strongly inhibited, and the inhibition degree is corresponding to the toxicity and concentration of the received toxic and harmful substances. For example, when nitrite, arsenite, ammonia, heavy metals, etc. are encountered, its luminescence process is rapidly inhibited. Therefore, the vibrio qinghaiensis Q67 is a freshwater luminous bacterium with stable luminescence and sensitivity to external toxic substances, and has important application value in environmental water quality detection. However, in the prior art, the suspension (working bacteria liquid) prepared on site by the luminous bacteria is directly adopted for toxicity detection, the on-site manual configuration is more time-consuming, and the number of active strains in the suspension is difficult to meet the requirement of detection standards.
In the prior art, the Chinese invention document CN106525822A provides a method for predicting the acute combined toxicity of three pesticides to luminous bacteria in order to solve the problems of large test workload, quantitative evaluation and lack of an acute combined toxicity effect prediction method of the existing toxicology acute combined toxicity evaluation technology, but the method cannot qualitatively judge the combined toxicity property (antagonism, independence, addition or synergy), and the quantitative prediction of the acute combined toxicity of the three pesticides on the luminous bacteria can be realized only by establishing a luminous bacteria relative luminous inhibition rate model based on the concentration of the three pesticides, so the method still needs to be improved.
Therefore, the prior art is directly used for carrying out acute toxicity test of water quality in the field, especially for detecting the combined toxicity effect of luminous bacteria on the composite pollutants, and the defects of equipment, bacterial liquid, long time consumption in the detection process, unstable test results and the like exist. In the prior art, devices such as on-line monitoring of toxicity test and portable toxicity analyzers are reported, and mainly relate to technologies such as on-line monitoring instruments prepared by utilizing luminous bacteria, portable water quality comprehensive toxicity analyzers, biological toxicity analyzers and biological early warning systems based on water quality monitoring, and the common characteristics of the technologies are as follows: the traditional chemiluminescent enzyme-labeled instrument is adopted, and the luminescent working bacteria liquid prepared on site is thawed by using the freeze-dried powder, is exposed in a water sample, and is used for carrying out on-line monitoring, acute toxicity testing or comprehensive toxicity monitoring. However, the defects that the test equipment is difficult to carry, the water sample to be tested needs to be pretreated, the working bacterial liquid needs to be prepared on site, the test equipment is large in size (an external power supply is usually needed), the test method is complicated, the time consumption is long, the influence of the quality of freeze-dried powder, the test environment conditions (temperature and cleanliness) and the like is easy to realize, the quick on-site combined toxicity detection under the field environment is difficult to realize, and the experimental result is difficult to ensure that the working bacterial liquid and the pretreatment process are not influenced and the like are overcome.
Therefore, in the method for testing the water quality acute toxicity test luminescent bacteria provided by the prior art, the suspension (working bacterial liquid) of the luminescent bacteria is directly exposed for toxicity detection, the test water sample needs to be pretreated, the test equipment needs to be externally connected with a power supply, so that the test method is complicated, the equipment is difficult to carry and work under the field condition, the rapid combined toxicity detection on the field site cannot be realized, the test result is also easily influenced by pretreatment, the quality of freeze-dried powder, the bacterial liquid re-dissolution process and the like, the batch and rapid detection of the rapid development of the water quality combined acute toxicity under the field condition cannot be realized, the accurate result is obtained, the requirements of the water quality toxicity emergency combined detection and the mechanical detection under the field condition are met, and the popularization and application are met in a large scale.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a combined acute toxicity test method for field water quality on-site detection, aiming at the defects that the combined toxicity effect detection of the combined pollutants in a water sample is needed, the preparation of detection, the detection process and the detection instrument are greatly simplified through the cooperation of prefabricated luminous bacteria gel beads and a portable handheld water quality acute toxicity detector, the water sample detection of one batch can be completed within 10 minutes at maximum after the detection result is obtained from the preparation of a detection material, the defects that the test equipment cannot be carried, an external power supply is needed, the water sample is needed to be pretreated, the bacteria liquid is needed to be prepared on site and the like, the detection material is more, the method is complicated, the rapid and batch combined toxicity detection under the field on-site condition cannot be realized, and the number of active bacterial strains in the bacteria liquid after the bacteria agent is re-dissolved is difficult to ensure to meet the standard requirement, the experimental result is easily influenced by factors such as unstable number of the active bacterial strains, manual influence factors of operators and the like, and the requirements of large-scale popularization and application and popularization can be met.
The technical scheme provided by the invention for solving the problems is as follows:
a combined acute toxicity test method for field water quality on-site detection comprises the following steps:
s1: preparing a portable handheld water quality acute toxicity detector, wherein the detector comprises a shell, a display panel, a bioluminescence photometer and a detection bin, wherein the display panel, the bioluminescence photometer and the detection bin are arranged in the shell, a detection pore plate is arranged in the detection bin, and a plurality of detection holes are formed in the detection pore plate; the volume of each detection hole arranged on the detection hole plate is 200 mu L;
s2: preparing a plurality of luminous fungus gel beads in batches in advance, ensuring that the number of active luminous strains meeting the detection standard requirement in the coating of each luminous fungus gel bead, and freezing and preserving; the volume of the single luminous fungus gel beads is 50-60 mu LThe number of active luminous strains in the content of luminous fungus gel beads is not less than 2×10 on average 5 The method comprises the steps of carrying out a first treatment on the surface of the The pore diameter of the coating is 300-500 nm, and the pore diameter of the hydrophilic network structure inside the coating is 500-800 nm;
s3: carrying a portable handheld water quality acute toxicity detector and frozen batch luminous fungus gel beads to a field water quality detection site, and collecting or preparing a water sample to be detected on the site, wherein the water sample to be detected comprises at least two toxic substances (such as inorganic substances and organic substances with different molecular diameters);
S4: taking out frozen luminous fungus gel beads in batches, naturally thawing at normal temperature, and recovering activity of luminous fungus in the capsule of each luminous fungus gel bead;
s5: respectively injecting a water sample to be detected into a detection pore plate, then putting a luminous fungus gel bead into each detection pore, and then putting the detection pore plate together with the water sample to be detected and the luminous fungus gel beads into a detection bin in a horizontal state;
s6: in 30 minutes, the toxic substances with small molecular diameters (such as inorganic substances) in the water sample to be detected pass through the coating to reach the inside of the coating, influence the fluorescence of the luminous strain, form the fluorescence change rate in a first time period, and then the organic toxic substances with large molecular diameters (such as inorganic substances) also reach the inside of the coating, interact with the toxic substances with small molecular diameters which reach in advance, and jointly influence the luminous strain to form the fluorescence change rate in a second time period; using a bioluminescence photometer in a portable handheld water quality acute toxicity detector to respectively acquire and record fluorescence change data of luminous strains in each detection hole according to time sequence, and obtaining the luminous inhibition rate of one toxic substance in the object to be detected in each detection hole to the luminous strains in the luminous fungus gel beads and the luminous inhibition rate of the two toxic substances together to the luminous strains in the luminous fungus gel beads after calculation, thereby obtaining combined acute toxicity data after the combined action of the two toxic substances, and the interaction type of the two toxic substances belongs to synergistic action, additive action or antagonistic action; and then respectively calculating to obtain concentration data of two corresponding toxic substances in the target objects to be detected in each detection hole, recording and outputting the concentration data through a display panel, finishing the on-site combined acute toxicity test of water quality rapid detection under the field condition of one batch, and simultaneously finishing the evaluation of the toxic effect type when the two toxic substances are subjected to combined pollution.
S7: after a batch of tests are completed, the detection pore plate is taken out from the detection bin, the completely detected water sample and the luminous fungus gel beads are recovered into a specified container, and the detection pore plate is emptied;
s8: and repeating the steps S4-S7 to finish the combined acute toxicity detection of the water samples to be detected in the subsequent batches until the detection of all the water samples to be detected is finished.
The toxic substances with small molecular diameter comprise inorganic heavy metal ions such as chromium, mercury, zinc, copper, lead, cadmium, arsenic and the like; the toxic substances with large molecular diameters comprise pesticides, antibiotics, chlorophenols, phenols and phthalate esters of organic substances.
Compared with the prior art, the combined acute toxicity test method for field water quality on-site detection has the beneficial effects that:
1. the combined acute toxicity test method for field water quality on-site detection provided by the invention is characterized in that the two are closely matched by prefabricating luminous bacteria gel beads and improving the structure of a portable handheld water quality acute toxicity detector, and the binary combined acute toxicity of the composite pollutant is detected by utilizing the coating structure of the luminous bacteria gel beads (different in permeability of two types of toxic substances with different molecular weights) and the specific combined action type of the two types of toxic substances can be detected; the method can realize rapid, efficient, accurate and batch water sample combined toxicity detection under the field environment condition, greatly simplify detection preparation, detection process and detection instrument, and complete water sample detection of one batch within 10 minutes from preparation of detection materials to obtaining of detection results, which is only 20-30% of the prior art.
2. The combined acute toxicity test method for field water quality on-site detection improves the structure of a portable handheld water quality acute toxicity detector in the prior art, uses a self-powered battery, removes a reaction test tube which cannot support quantitative constant-volume luminous fungus gel beads and needs to be sealed in detection, changes a porous detection pore plate (such as 18 holes) with an open structure, does not need to be sealed in the detection process (only needs to be placed horizontally), and is matched with the luminous fungus gel beads prepared in batches in advance, one hole is used for one bead, so that the operations such as putting in, detecting and taking out in the batch detection process are very convenient, a water sample does not need to be pretreated, the luminous fungus gel beads can be used after being naturally thawed, the quantity of luminous strains is enough, and mixed bacteria in a field environment are avoided, so that the stability and the accuracy of a detection result are improved while the operation steps are simplified. According to the method, through the difference of interaction (permeability and penetrability) between different toxic molecules and the luminous fungus gel beads, the combined toxic effect (antagonism, independence, addition or synergy) between different types of toxic substances can be qualitatively judged in a one-time detection process, a luminous fungus relative luminous inhibition rate model based on the concentration of toxic substances such as pesticides and the like is not required to be established, and quantitative prediction of the combined action of various toxic substances (pesticides or other organic substances and inorganic substances such as heavy metals) on the acute combined toxicity of the luminous fungus can be realized through one-time detection, so that the method has the advantages of larger breakthrough, simplicity and convenience in operation and capability of expanding the applicable range of combined toxic effect detection substances, and can be used for simultaneously detecting the combined toxic effect between different types of organic substances and inorganic substances, and corresponding toxic effect and quantitative concentration can be obtained through one-time detection, so that the detection operation steps are greatly simplified, and the detection time and the detection consumable are saved.
3. According to the combined acute toxicity test method for field water quality on-site detection, the luminous fungus gel beads prepared in batches in advance under laboratory conditions are adopted to replace the traditional luminous fungus liquid (working fungus liquid) prepared on site, so that on one hand, the number of active luminous strains meeting standard requirements in an envelope can be ensured, and in addition, the links of re-dissolving, stirring and the like under the field conditions can be omitted, and the entry of mixed bacteria in the environment can be avoided; thirdly, the luminous fungus gel beads are designed for constant volume (50-60 mu L) and quantitative (living fungus number), the volume of each luminous fungus gel bead and the number of luminous strains with content activity are controlled during preparation, the volume of water samples (200 mu L) of detection holes are completely matched with the requirements of detection standards, when in field detection, an independent luminous fungus gel bead which is naturally thawed for 5 minutes and returns to normal temperature is correspondingly placed in each detection hole, the luminous strains in the gel beads can restore activity, water samples containing objects to be detected are injected into each detection hole within 30 minutes, the luminous inhibition rate of the objects to be detected on the luminous strains in the luminous fungus gel beads is automatically observed by an instrument, the concentration of the objects to be detected can be obtained after calculation, the operation is convenient and quick, a batch (when an 18-hole detection plate is adopted, a batch of 18 water samples can be detected) can be completed within 10 minutes at most, and the portable handheld water quality acute toxicity detector in the prior art can detect 3 water samples each batch, and the detection efficiency of the portable handheld water quality acute toxicity detector in batches is more than 10 times than the prior art.
4. According to the combined acute toxicity test method for field water quality on-site detection, which is provided by the invention, aiming at a plurality of defects of working bacteria liquid, sample pretreatment and the like in the prior art, by matching components, proportion, structure and preparation technology of the luminous bacteria gel beads, embedding luminous bacteria by adopting an immobilization technology, embedding a required number of active luminous strains in a specific material to form mutually independent luminous bacteria gel beads with specific volume, and then integrally freezing and transporting the luminous bacteria gel beads for direct acute toxicity test of a water quality sample under field conditions, wherein each detection hole is provided with a naturally thawed luminous bacteria gel bead for detection, a water sample is not required to be pretreated, so that the defects that the number of test materials is large, the test method is complicated, the field on-site detection environment cannot be adapted, the number of active bacterial strains in the bacteria liquid after the bacterial agent is re-dissolved is difficult to ensure to meet the standard requirements, the experimental result is easily influenced by external factors, the number of the bacterial strains is unstable, operators are artificial and the like are overcome; the luminous bacteria gel beads prepared by standardization are matched with the improved portable handheld water quality acute toxicity detector, so that the luminous bacteria gel beads can meet the environmental conditions of a field site, and the operation method is simple and convenient and can be standardized, thereby meeting the requirements of large-scale and large-scale popularization and application and popularization.
5. Aiming at the requirements of rapid and accurate preparation of detection materials in the field or on site (under non-laboratory conditions), the invention embeds the luminous bacteria by adopting an active immobilization technology through matching the strains, components, proportions, structures and preparation processes of prefabricated luminous bacteria gel beads, embeds the active luminous strains meeting the requirement in specific materials to form mutually independent luminous bacteria gel beads with specific volumes, and freezes and transports the luminous bacteria gel beads as a whole for direct acute toxicity test of water quality samples under the field conditions; according to the detection requirement of the normal volume (200 mu L) of each detection hole, specially designing and preparing luminous fungus gel beads which are moderate in size, portable and up to standard in viable bacteria concentration after reactivation; the preparation method has the advantages that the proper luminous strains and film forming materials are selected, the luminous strains and film forming materials are prepared into finished products in batches, and the finished products can be stored, carried and transported at low temperature, so that the problems of inaccurate detection results and the like caused by inaccurate concentration of prepared test bacteria due to influence of factors such as condition limitation and manual operation and the like in preparing bacterial liquid by using frozen bacterial powder on site are solved.
6. The luminous fungus gel beads prepared by the method can be prepared in batches under laboratory conditions, and then can be preserved and transported under-4 ℃ freezing conditions, so that the luminous strains can keep activity continuously inside the gel beads; after the gel beads are transported to a detection site, the gel beads are naturally thawed to normal temperature, most of luminous strains can restore activity automatically, and the number of effective bacteria and the volume of detection holes which are required to be ensured in each gel bead are comprehensively considered when the gel beads are prepared, so that the gel beads are designed into specific volumes, surplus bacterial strains are immobilized, one gel bead is placed in each detection hole after thawing, the requirements of the standard on the luminous bacteria concentration can be met, and the detection can be completed within 10-30 minutes.
7. The luminous fungus gel beads adopted by the invention are coated with calcium alginate films, and the pore diameters of the pores of the films are suitable, so that molecules of the tested toxic substances can smoothly pass through and quickly permeate into the gel beads to test, and meanwhile, the completeness of the appearance of the luminous fungus gel beads in the detection process can be ensured, and the luminous strains can be continuously restrained in the gel beads. According to SEM (electron microscope) scanning result analysis, the surface of the calcium alginate film has obvious holes, and the pore size is about 0.5 mu m; the internal structure of the content of the luminous fungus gel beads is a hydrophilic net structure filled by mixing calcium alginate and sodium alginate, the hydrophilic net structure has larger pore diameter, the molecular diameter of inorganic substances in a toxicity detection target to be detected is about 0.1-0.5 nm, and the molecular diameter of organic substances is about 100-220 nm, and the molecular diameters are far smaller than the pore sizes of a film and the content, so that the luminous fungus gel beads can pass through the film and the content, smoothly enter the luminous fungus gel beads, influence the activity of internal luminous strains and finish detection; however, the penetrability and the fluidity of the two materials are different, and a certain time difference exists between reaching the inside; the change of the luminous rate caused by the time difference can be applied to the analysis of the combined toxicity effect.
8. The invention also fully considers the relativity of the luminous process of the luminous bacteria in the luminous bacteria gel beads and the activity and physiological metabolism activity of cells, the luminous bacteria are taken as toxic test organisms, the luminous intensity of the luminous bacteria is kept constant under certain test conditions, the physiological activity of the cells is changed due to the toxic effect of pollutants, the bioluminescence intensity is weakened, and the degree of change is related to the concentration of the test objects in a certain range. Therefore, the invention can evaluate the toxicity (or combined toxicity) degree of the tested substances by measuring the reduction degree (fluorescence change rate) of the overall luminosity of the luminous bacteria in the luminous bacteria gel beads. The change of the luminous intensity can be measured by a luminous photometer, and the method has the advantages of less time consumption, high sensitivity, simple operation and accurate result.
9. The vibrio qinghaiensis adopted by the embodiment of the invention is the only nonpathogenic freshwater luminous bacteria, the freeze-dried powder of the vibrio qinghaiensis is safe and reliable in the transportation and use processes, and the waste bacterial liquid does not need special treatment and does not cause secondary pollution. The method of the invention uses a variant of Vibrio qinghaiensis Q67 (the original strain is provided by the national institute of university of Shandong's student's life) as an index organism for toxicity detection of aquatic organisms, and has the advantages of rapidness, simplicity and high sensitivity. The gel pellet prepared by the invention has the advantages of cheap and easily available raw materials, simpler conditions for preparing gel and culturing strains, and a simple method for rapidly detecting the toxicity of the pollutants in the water body sample on site can be designed based on the gel pellet. The water quality detection method for fixing the vibrio qinghaiensis Q67 is simple and easy to implement, the analytical instrument and the operation method used for experiments are simple and convenient, and the method can be widely applied to qualitative detection in non-professional fields.
10. Through practical tests, the invention adopts the prefabricated luminous bacterial gel and the improved portable handheld water quality acute toxicity detector, can be widely applied to various field on-site development water quality combined acute toxicity tests, can greatly reduce the types and the quantity of detection materials, simplify the test method, can realize on-site batch detection, and prevent the detection result from being influenced by the uncertainty processes such as water sample pretreatment, bacterial liquid redissolution and the like, has quick and convenient batch detection process and accurate combined toxicity detection result, breaks through the limitations of the existing similar products and technologies, and can meet the requirements of large-scale, large-scale and low-cost popularization and application.
Drawings
FIG. 1 is a schematic view of the appearance of a plurality of mutually independent luminous fungus gel beads in a water body according to an embodiment of the invention;
FIG. 2 is a schematic outline of a single luminescent bacterial gel bead in accordance with an embodiment of the present invention;
FIG. 3 is a schematic illustration of a preparation flow of luminescent bacterial gel beads according to an embodiment of the present invention;
FIG. 4 is a graph showing luminosity response of the luminous fungus gel beads according to the embodiment of the invention compared with that of the traditional luminous fungus liquid;
FIG. 5 is a schematic diagram showing the response results of the luminescent bacteria in the luminescent bacteria gel beads in the embodiment of the invention in the simulated lake water and potassium permanganate;
FIG. 6 is a schematic diagram showing experimental results of the durability of the response of the luminous bacteria in the luminous bacteria gel beads according to the embodiment of the invention;
FIG. 7 is a graph showing the inhibition ratio of phenol to light emission of light emitting bacterium Q67 according to the embodiment of the present invention;
FIG. 8 is a graph showing the inhibition ratio of mercury chloride to light emission of light emitting bacterium Q67 according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Example 1
Referring to fig. 1-2, the combined acute toxicity test method for field water quality field detection provided by the embodiment of the invention can be applied to batch combined toxicity (binary combined acute toxicity) detection of composite water samples of different river segments of polluted river collected in field under field conditions so as to quickly find out the types, concentrations and change trends of pollutants in each segment of river and provide a data basis for subsequent tracing, and comprises the following steps:
S1: preparing a portable handheld water quality acute toxicity detector, wherein the detector comprises a shell, a display panel, a bioluminescence photometer and a detection bin, wherein the display panel, the bioluminescence photometer and the detection bin are arranged in the shell, a detection pore plate is arranged in the detection bin, a plurality of detection holes are formed in the detection pore plate, and the volume of each detection hole is 200 mu L; the detector adopts a self-powered (lithium battery) without an external power supply; the embodiment adopts a portable handheld water quality acute toxicity detector which is designed by improving the prior art, removes a reaction tube which cannot support quantitative constant volume luminous fungus gel beads, is complex to operate and needs to be sealed during detection, and is replaced by a porous detection pore plate with an open structure.
In the embodiment, the biological luminescence photometer is improved on the basis of a handheld luminescence bacterial toxicity detector with a commercial model, and the technical specification of the biological luminescence photometer adopted by the detector is as follows: the spectral range can be detected, and the range is 320 nm-1000 nm; detecting the result range, namely, 0-65535 RLU; the fastest detection time is 5min; relative luminous intensity, 0-200%; ATP test and detection pore plate test can be performed; the detection bin and the detection pore plate are square in design, and the outline and the volume are mutually matched; the detection bin is also provided with a bin cover which can be pivoted on one side and is opened and closed in a rotating way when the detection pore plate is added or taken out, and the detection pore plate is closed in a detection or non-use way. The detector is also provided with a calculation program by adopting a conventional technology, and can automatically calculate the concentration of the target object to be detected in each detection hole according to the collected luminous signals, automatically record and summarize, and display the concentration on a display panel without human intervention;
S2: preparing a plurality of luminous fungus gel beads in batches in advance, ensuring that the number of active luminous strains meeting the detection standard requirement in the coating of each luminous fungus gel bead, and freezing and preserving; the volume of the single luminous fungus gel beads is 50-60 mu L, and the number of active luminous strains in the content of the luminous fungus gel beads is not less than 2 multiplied by 10 on average 5 Matching the capacity of the detection hole in the step S1; the pore diameter of the coating is 300-500 nm, the pore diameter of a hydrophilic network structure in the coating is 500-800 nm, and the penetration and fluidity of two toxic substances are obviously different due to the mutually combined structure of the membrane and the network; specifically, the following steps M1-M4 are adopted;
s3: carrying a portable handheld water quality acute toxicity detector and frozen batch luminous fungus gel beads to a field water quality detection site, collecting or preparing a water sample to be detected on the site, wherein the water sample to be detected comprises at least two toxic substances (such as inorganic substances and organic substances with different molecular diameters or two substances with different molecular weights) with different molecular diameters, so as to perform binary combined acute toxicity detection on the two toxic substances;
s4: taking out frozen luminous fungus gel beads in batches, naturally thawing at normal temperature, and recovering activity of luminous fungus in the capsule of each luminous fungus gel bead; in actual operation, the luminous fungus gel beads with sufficient quantity can be thawed in batch in advance at one time according to the quantity of water samples to be detected, and detection of a plurality of batches is supported;
S5: respectively injecting each water sample to be detected in a first batch into a detection pore plate, then putting a luminous fungus gel bead into each detection pore plate, putting the detection pore plate together with the water sample to be detected and the luminous fungus gel beads into a detection bin in a horizontal state, closing a bin door of the detection bin, and keeping the detector horizontally placed all the time in the detection process;
s6: in 30 minutes, the toxic substances with small molecular diameters (such as inorganic substances) in the water sample to be detected pass through the coating to reach the inside of the coating, influence the fluorescence of the luminous strain, form the fluorescence change rate in a first time period (15-60 s), and then the toxic substances with large molecular diameters (such as inorganic substances) also reach the inside of the coating to interact with the toxic substances with small molecular diameters which reach in advance to jointly influence the luminous strain and form the fluorescence change rate in a second time period (61-300 s); using a bioluminescence photometer in a portable handheld water quality acute toxicity detector to respectively acquire and record fluorescence change data of luminous strains in each detection hole according to time sequence, and obtaining the luminous inhibition rate of one toxic substance in the object to be detected in each detection hole to the luminous strains in the luminous fungus gel beads and the luminous inhibition rate of the two toxic substances together to the luminous strains in the luminous fungus gel beads after calculation, thereby obtaining combined acute toxicity data after the combined action of the two toxic substances, and the interaction type of the two toxic substances belongs to synergistic action, additive action or antagonistic action; respectively calculating to obtain concentration data of two corresponding toxic substances in the target object to be detected in each detection hole, recording and outputting the concentration data through a display panel, finishing on-site acute toxicity test (namely binary combined acute toxicity detection) for rapidly detecting water quality under a batch of field conditions, and simultaneously finishing evaluation of toxic effect types when two (or two types of) toxic substances are subjected to combined pollution;
S7: after a batch of tests are completed, the detection pore plate is taken out from the detection bin, the completely detected water sample and the luminous fungus gel beads are recovered into a specified container, and the detection pore plate is emptied;
s8: and repeating the steps S4-S7 to finish the combined acute toxicity detection of the water samples to be detected in the subsequent batches until the detection of all the water samples to be detected and the evaluation of the toxic effect type when the two toxic substances are subjected to combined pollution are finished.
The toxic substances with small molecular diameter comprise inorganic heavy metal ions such as chromium, mercury, zinc, copper, lead, cadmium, arsenic and the like, and the molecular diameter is generally 0.1-0.5 nm; the toxic substances with large molecular diameters comprise pesticides with organic substances, antibiotics, chlorophenols, phenols, phthalate compounds and the like, and the molecular diameters of the toxic substances are generally 100-220 nm. Because the molecular size and the shape of two toxic substances (or two toxic substances with different molecular weights in the same type of substances) in the two types of toxic substances are different, inorganic toxic substance molecules in the water sample penetrate through the coating before organic toxic substances penetrate through the coating and reach the inside of the coating, and the time difference of reaching the inside is about 15-60 s through testing; specific factors influencing the time difference are complex, mainly the molecular size and shape (penetrability) of toxic substances, the compatibility of toxic substance molecules with envelope molecules (calcium alginate), liquid sodium alginate molecules in the content (intermolecular force), environmental temperature and the like.
In the embodiment of the invention, a single batch of portable handheld water quality acute toxicity detectors can detect 18 water samples, and the time is about 10 minutes; the second batch takes only 5-6 minutes (the time for naturally thawing the luminous fungus gel beads is saved), and the batch detection efficiency is 10-20 times of that of the prior art.
Referring to fig. 1-2, the luminous fungus gel bead provided in this embodiment is a solid spherical gel bead with complete shape and smooth surface, and the gel bead includes an envelope and a content completely covered by the envelope; the content is gel liquid including luminous bacteria liquid, and the coating is a film formed by rapidly carrying out gel reaction on the contact surface after the gel liquid contacts with film forming reaction solution; the method comprises the steps that a plurality of luminous strains in luminous bacterial liquid are fixed in gel beads by an envelope, and when a target object to be detected passes through the envelope and reaches the inside of the gel beads, the activity of the luminous strains in the envelope is influenced based on the toxicity concentration of the target object to be detected; the luminous fungus gel beads keep complete shape in the whole water sample detection process, and the coating continuously restrains luminous strains in the film.
The volume of each single independent luminous fungus gel bead is 50-60 mu L, and the number of active luminous strains in the content is not less than 2 multiplied by 10 on average 5 The method comprises the steps of carrying out a first treatment on the surface of the The luminous strain is as follows: vibrio qinghaiensis Q67Vibrio qinghaiensis sp.nov.Q67(hereinafter referred to as Q67), or Photobacterium brightlyPhotobacterium phosphoreum T3；Or Photobacterium brightens ATCC11040, or Vibrio fischeri. Wherein, the original strain of the vibrio qinghaiensis Q67 is provided by the university of eastern China student's life sciences college, and other original strains can be obtained through market purchase.
Referring to fig. 3, the preparation method of the luminous fungus gel beads comprises the following steps:
m1, culturing luminous strains in batches or redissolving luminous fungus freeze-dried powder in a laboratory environment (or a clean factory environment) to obtain luminous fungus liquid;
wherein, the step of culturing the luminous strains in batches comprises the following steps:
m11: preparing a culture medium: preparing the solution with pH of 9 according to the proportion, and adding the following components KH in mass ratio into each milliliter of the solution 2 PO 4 （13.6 mg）、Na 2 HPO 4 ·12H 2 O（35.8 mg）、MgSO 4 ·7H 2 O（250.0 mg）、MgCl 2 ·6H 2 O（610.0 mg）、CaCl 2 （33.0 mg）、NaHCO 3 (1340 mg), naCl (1540 mg), yeast extract (5000 mg), tryptone (5000 mg) and glycerol (3000 mg) to form a liquid medium, and adding 2wt% agar to the liquid medium to form a solid medium;
m12: culturing in a culture medium: firstly, placing the luminous strain on the inclined plane of a solid culture medium, and culturing for 24 hours at the temperature of 22 ℃ in a biochemical incubator; inoculating to a triangular flask filled with sterile liquid culture medium, placing into a constant temperature shake incubator, shake culturing at 22deg.C under 160 r/min, and taking out;
M13: and detecting by an enzyme-labeled instrument, and obtaining luminous bacterial liquid for standby when the luminosity is stabilized above 1000000 and RU.
M2, preparing gel liquid including luminous bacterial liquid to obtain luminous bacterial gel liquid with proper luminous bacterial strain concentration content;
m21: preparation of gel base solution
Weighing polyvinyl alcohol and sodium alginate by using an analytical balance, placing the polyvinyl alcohol and sodium alginate into a beaker, adding ultrapure water, heating while continuously stirring, heating until the polyvinyl alcohol and the sodium alginate are completely dissolved, taking out, standing, cooling, and placing the cooled polyvinyl alcohol and sodium alginate into a refrigerating chamber of a refrigerator at 4 ℃ for refrigerating until bubbles disappear to obtain gel base liquid for later use;
m22: preparation of luminous bacterial liquid
And (3) preparing the luminous bacterial liquid with the proper luminous bacterial strain concentration content prepared in the step (M1) or the luminous bacterial liquid with the proper luminous bacterial strain concentration content prepared by redissolving the luminous bacterial lyophilized powder, and the gel base liquid prepared in the step (M21) according to the following steps of 1:1 to obtain the luminous strain gel liquid with proper concentration and content of luminous strain.
The step M22 further comprises a step M221 of adding a resuscitation promoter liquid:
adding glucose into the luminous bacterial liquid prepared in the step M1 to enable the concentration of the glucose in the bacterial liquid to reach 5wt%; or adding sodium chloride to make the concentration of sodium chloride in the bacterial liquid reach 0.8wt%, and then mixing with the gel base liquid prepared in the step M21 according to the following formula 1:1 to obtain the luminous strain gel liquid with proper concentration and content of luminous strain.
According to the embodiment, the reviving rate of the luminous bacteria is improved when the gel beads are thawed by supplementing a reviving promoter (glucose or NaCl) into the luminous bacteria liquid, so that the number of active luminous bacteria in each gel bead is ensured, and the standard requirement can be met after reviving; the reviving solution of the freeze-dried powder of the vibrio qinghaiensis is 5wt% of glucose fusion or 0.8wt% of sodium chloride solution.
M3, preparing a film forming reaction solution, namely dripping the luminous strain gel solution into the film forming reaction solution according to the volume range of 50-60 mu L per drop, wherein the film forming reaction solution rapidly forms a coating on the surface of the drop of the luminous strain gel solution, the coating completely coats and fixes the luminous strain gel solution in spherical gel beads, and the number of active luminous strains in the gel beads is not less than 2 multiplied by 10 on average 5 Obtaining an independent luminous fungus gel bead after dripping once; the method specifically comprises the following steps:
m31: preparing a film forming reaction solution: preparing a calcium chloride aqueous solution with the concentration of 20-25 mg/mL as a film forming reaction solution;
m32: sucking the luminous strain gel liquid prepared in the step M2 by using a syringe, then slowly dripping the luminous strain gel liquid in the syringe into a film forming reaction solution according to the volume range of 50-60 mu L per drop, wherein the film forming reaction solution rapidly forms a coating on the surface of the drop of the dripped luminous strain gel liquid, particularly sodium alginate and calcium chloride rapidly undergo a gel reaction, and the surface of the dripped drop is coagulated into the coating, so that the dripped gel drop forms a solid spherical luminous fungus gel bead with complete shape and smooth surface; the gel liquid of luminous strain is completely coated and fixed in spherical gel beads, and the number of active luminous strains in the gel beads is not less than 2×10 5 Obtaining an independent luminous fungus gel bead after dripping once;
m33, dripping the luminous strain gel liquid sucked into the syringe for multiple times according to the same volume, so that each drop of luminous strain gel liquid is slowly dripped into the film forming reaction solution; after the syringe is dripped, extracting again, and sequentially repeating the operation until all the prepared luminous strain gel liquid is dripped, and preparing a plurality of mutually independent luminous strain gel beads in batches; the pore diameter of the gel liquid coating of the luminous strain is 300-500 nm, the pore diameter of a hydrophilic network structure in the coating is 500-800 nm, and the structure that the membrane and the network are combined with each other ensures that the penetrability and the fluidity of two toxic substances are obviously different, and the structural characteristic can support the combined toxicity detection of a plurality of toxic substances with different molecular weights;
when a 2ml syringe is adopted in the embodiment, about 35 luminous bacteria immobilized gel beads with complete shapes can be prepared by mixing gel bacterial liquid with 2ml per syringe.
M4: repeating the step M3, sucking the luminous fungus gel liquid for a plurality of times by using a needle cylinder, slowly dripping the luminous fungus gel liquid into the film forming reaction solution to obtain luminous fungus gel beads, and preparing a plurality of mutually independent luminous fungus gel beads in batches until the luminous strain gel liquid prepared in the step M2 is completely dripped; the preparation method comprises the steps of placing a plurality of luminous fungus gel beads prepared in batches, the rest film-forming reaction solution and a container in a refrigerator as a whole, freezing and maintaining at-4 ℃, transporting, taking out the luminous fungus gel beads in batches again as a whole under non-laboratory conditions when in use, taking enough luminous fungus gel beads in batches for use, naturally melting for 5 minutes on site, and recovering to normal temperature, wherein each strain can recover activity at the moment, can replace the traditional working fungus liquid, and is integrally applied to water quality acute toxicity detection under field conditions.
Referring to fig. 4-8, the combined acute toxicity test method for field water quality on-site detection provided by the embodiment adopts the luminous fungus gel beads to replace the traditional luminous fungus liquid, ensures the number of active luminous strains meeting the standard requirements in the coating, and performs field water quality on-site acute toxicity test; placing an independent luminous fungus gel bead which is restored to normal temperature in each detection hole, injecting a target object to be detected into each detection hole within 30 minutes after naturally thawing for 5 minutes, and observing the luminous inhibition rate of the target object to be detected on luminous strains in the luminous fungus gel beads to obtain the concentration of the target object to be detected; the volume of each detection hole is 200 mu L, and the standard detection hole plate which is most commonly used at present is adopted; the luminous fungus gel beads are completely matched with the volume of the detection holes, so that the number of active luminous strains for detection is ensured to meet or exceed the number requirement of detection standards.
In the embodiment, the reviving rate of the luminous bacteria can be improved when the luminous bacteria gel beads are naturally thawed by supplementing the revival promoting liquid (glucose or NaCl), the number of the active luminous bacteria in each gel bead is ensured, and the number of the revived small part and most strains which cannot revive can meet the standard requirement. The freeze-dried powder resuscitation liquid of the vibrio qinghaiensis Q67 adopted by the embodiment of the invention is 5% glucose fusion or 0.8% sodium chloride solution.
In order to determine the optimal time for testing after resuscitating the lyophilized powder, the embodiment determines the luminous intensity at different times after resuscitating, and as can be seen from the data in table 1 (the lyophilized powder resuscitating time) below, the luminous intensity of the luminescent bacteria can be kept stable after 5 minutes of natural thawing.
TABLE 1
The embodiment adopts various technical means, can ensure the concentration of viable bacteria in each luminous fungus gel bead during preparation and the number of viable bacteria in each luminous fungus gel bead after recovery, and can ensure that the concentration of viable bacteria can reach the minimum requirement of detection standard (the number of viable bacteria required by 200 mu L of detection sample of a detection hole). The Vibrio qinghaiensis Q67 adopted by the invention can still grow normally at 4 ℃, has higher reviving rate, and is generally higher than 99% under the condition of supplementing resuscitation promoting liquid; in the case where the resuscitation inducing liquid is not supplemented, the concentration is usually higher than 96%.
The luminous fungus gel beads prepared in the embodiment can be prepared in batches under laboratory conditions, and then can be preserved and transported under the freezing condition of-4 ℃ in a refrigerator, and the luminous strain can continuously maintain the activity in the process; after the gel beads are transported to a detection site, the gel beads are naturally thawed to normal temperature, most of luminous strains can restore the activity by themselves, and as the number of the effective bacteria which can be revived in each gel bead is repeatedly considered when the gel beads are prepared and a sufficient number of the strains are provided when the gel beads are immobilized and embedded, each detection hole only needs to be provided with one gel bead, wherein the number of the revived strains can meet the requirement of the standard on the concentration of the luminous bacteria, and the detection personnel can finish the detection within 10-30 minutes.
In addition, through practical tests, the pore diameter of the pores of the gel bead coating (calcium alginate film) is proper, so that molecules of two types of tested toxic substances can smoothly pass through and quickly permeate into the gel beads, the toxic concentration of the two types of toxic substances can be detected, and the penetrability (penetration speed) and the flowability (penetration speed) of toxic substances with different molecular weights and molecular shapes (physicochemical properties) are different.
According to the embodiment of the invention, aiming at the requirements of quick and accurate preparation of detection materials in the field or on-site (under non-laboratory conditions), gel beads with moderate size, portability and standard concentration of live bacteria after reactivation are designed and prepared according to the detection requirements of the volume (200 mu L) of each detection hole, and proper luminous fungus beads and film forming materials are selected to be batched and prepared into finished products, so that the problems of inaccurate detection results and the like caused by condition limitation, manual operation and other factors when the frozen fungus powder is used for preparing fungus liquid on site are overcome; and then through the cooperative improvement and matching of the handheld detector, the detection pore plate and the luminous fungus gel beads, the detection equipment, the detection materials and the detection operation steps are greatly simplified, the environmental condition adaptability and the detection efficiency are improved, the batch, rapid and combined detection on the water quality toxicity under the field condition can be realized, and the combined toxicity detection result with good stability and high accuracy can be obtained.
Example 2
The embodiment of the invention provides a combined acute toxicity test method for field water quality on-site detection, which is based on the embodiment 1, and specifically provides a luminous fungus gel bead prepared by adopting vibrio qinghaiensis Q67 luminous fungus, and the combined acute toxicity test method for field water quality on-site detection is carried out, wherein the preparation method of the gel bead comprises the following steps:
preparing a vibrio qinghaiensis Q67 luminous bacteria culture medium:
(1) The formula of the liquid culture medium of the Vibrio qinghaiensis Q67 luminous bacteria is shown in the following table 2:
TABLE 2
(2) The formula of the solid culture medium of the vibrio qinghaiensis Q67 luminous bacteria comprises the following components: agar was added to the liquid medium composition in an amount of 2 wt%.
Culturing luminous bacteria:
regulating pH of the prepared culture medium to 9.0, subpackaging the liquid culture medium into 250 mL triangular bottles, sterilizing at high temperature of 121 ℃ for 20 minutes, pouring 15-20mL of culture solution into a culture dish after sterilizing, and preparing a culture medium plate before cooling the solid culture medium, wherein the culture medium is required to be refrigerated for standby. The vibrio qinghaiensis Q67 slant strain kept by cold storage at 4 ℃ is transferred to a fresh slant culture medium in a sterile workbench, and is placed in a biochemical incubator at 22 ℃ for culturing for 24 hours (the luminescence is observed in a darkroom), so that the strain can grow normally and the growth condition is good. Selecting a colony which grows well on a fresh inclined plane culture medium, inoculating the colony into a triangular flask filled with a 25 mL sterile liquid culture medium, putting the triangular flask into a constant-temperature shaking incubator, performing shaking culture at 22 ℃ and 160 r/min, taking out the colony after 18 h culture, and stabilizing the luminosity detected by an enzyme-labeling instrument to be more than 1000000 RLU for the next experimental operation.
(III) preparation of gel solution
10g of polyvinyl alcohol and 1g of sodium alginate are weighed respectively in an analysis day, put into a 250mL beaker, added with 100mL of ultrapure water, heated by an electric furnace, continuously stirred at the same time, heated for 10min until the polyvinyl alcohol and the sodium alginate are completely dissolved, taken out of the beaker, the polyvinyl alcohol and the sodium alginate solution are placed at room temperature, cooled and put into a refrigerator refrigerating chamber at 4 ℃ until bubbles disappear for later use.
(IV) preparation of gel beads for fixing luminous bacteria
Taking 50mL of the Vibrio qinghaiensis Q67 luminous bacterial liquid cultured at 22 ℃ and 160r/min, centrifuging for 10min at 2000r/min, and re-dissolving to 2mL by using simulated lake water; taking 1ml of gel liquid and 1ml of re-dissolved luminous bacteria liquid, uniformly mixing, sucking the mixed liquid by using a syringe, and dripping the mixed liquid into a calcium chloride solution according to an equal volume to form spherical gel beads with complete and smooth shapes, wherein 2ml of mixed gel bacteria liquid can be used for preparing 35 independent luminous bacteria immobilized gel beads with complete shapes, namely luminous bacteria gel beads.
Example 3
The combined acute toxicity test method for field water quality on-site detection provided by the embodiment of the invention adopts the luminous fungus gel beads prepared in the embodiment 2, and specifically compares the luminosity response of the luminous fungus gel beads in the acute toxicity test application of field water quality on-site detection with the luminosity response of the luminosity test directly carried out by adopting the traditional luminous fungus liquid.
In the embodiment, a potassium dichromate (reference substance) solution serving as a target to be detected is used as a positive control, and the prepared luminous bacteria gel beads (namely luminous bacteria immobilized gel beads) and luminous bacteria liquid (working bacteria liquid) prepared by a traditional method are used for respectively carrying out acute toxicity tests, and comparing and analyzing test results. The concentration gradient of the positive control potassium dichromate solution is 0.01mg/L,0.1mg/L,1mg/L,10mg/L and 100mg/L respectively.
The luminous response comparison step of the luminous fungus liquid direct test and luminous fungus gel bead test comprises the following steps:
and (3) directly testing the luminous fungus liquid: taking 96-well plates, adding 20 mu L of bacterial liquid into each well in rows A-F and columns 1-3, wherein rows A-F are blank, 0.01mg/L of potassium dichromate solution, 0.1mg/L of potassium dichromate solution, 1mg/L of potassium dichromate solution, 10mg/L of potassium dichromate solution and 100mg/L of potassium dichromate solution, and adding 190 mu L of each well, and setting 3 parallel wells.
And (3) testing luminous fungus gel beads: on a 96-well plate, one luminous fungus gel bead is added to each well in rows A-F and columns 4-6, wherein the rows A-F are respectively blank, 0.01mg/L potassium dichromate solution, 0.1mg/L potassium dichromate solution, 1mg/L potassium dichromate solution, 10mg/L potassium dichromate solution and 100mg/L potassium dichromate solution, 190 mu L are respectively added, and 3 parallel rows are arranged.
Referring to FIG. 4, a graph of luminosity response of a luminophor gel bead versus a conventional luminophor liquid is shown. FIG. 5 is a schematic diagram showing the response results of the luminescent bacteria in the luminescent bacteria gel beads in the embodiment of the invention in the simulated lake water and potassium permanganate.
As can be seen from the comparison results of the present example, the conventional luminescent bacteria liquid and the luminescent bacteria gel beads of the present example were contacted with the reference substance at the same time respectively, and after 15 minutes, the luminescent bacteria liquid in the 96 well plate and the luminescent bacteria in the luminescent bacteria gel beads were measured with the enzyme-labeled instrument, and the luminosity of the luminescent bacteria was measured once every one hour. The test result shows that the luminous intensity of the luminous fungus gel bead test has a trend of rising, and the luminous fungus gel bead material can delay the contact between the luminous strain and the reference object for a short time at the beginning, so that the luminous intensity has a trend of rising. However, in the whole, the luminous intensity peak value of the luminous bacteria detected in two ways is basically consistent with the effective time of the luminous bacteria, so that the luminous bacteria gel beads provided by the invention have the advantage that the luminous response result in the detection process is superior to that of the traditional working bacteria liquid.
Example 4
The combined acute toxicity test method for field water quality on-site detection provided by the embodiment of the invention adopts the luminous fungus gel beads prepared in the embodiment 2, and tests luminous fungus in the luminous fungus gel beads on the basis of the embodiment 3, and the response durability in the acute toxicity test process of field water quality on-site detection is realized. The method comprises the following specific steps:
The luminous fungus gel beads prepared in the calcium chloride solution are taken out every 1h and put into a 96-well plate to be respectively contacted with 1mg/L potassium dichromate solution and blank (simulated lake water) solution for 15min, and then the luminosity is measured. And (3) placing the prepared luminous fungus gel bead luminous fungus into a 96-well plate for 15min, and then placing into an enzyme-labeled instrument for luminosity detection. The luminous ability of the luminous bacteria in the luminous bacteria gel bead ball is kept at a higher level in the first 1h, and the luminous ability of the luminous bacteria gel bead ball is weakened after more than 1 h.
Referring to FIG. 6, a schematic diagram of the experimental results of the durability of the luminophor response in the luminophor gel beads is shown. Therefore, the luminous fungus gel beads provided by the invention have response durability superior to that of working fungus liquid in the prior art.
The luminous fungus gel beads and the water quality detection method provided by the invention also solve the problem of convenience in batch preparation and carrying of freshwater luminous strains through preparation in batches in advance, then freezing preservation and transportation.
In the prior art, working bacterial liquid is directly put into a water sample to be detected, so that the accurate number of active luminous strains can reach the standard requirement, and meanwhile, the direct contact of the strains with the water sample can cause unstable detection and accelerated inactivation of the strains; in the preparation process, the volume and the size of the immobilized bacteria beads can be accurately controlled, and the luminous bacteria gel beads prepared in batches have good uniformity and small difference, and the stability of the detection result is not affected; the raw materials used for preparing the luminous fungus gel beads in advance are cheap and easy to obtain, the cost for batch immobilized preparation and use is low, and the conditions are mild; the luminous fungus gel beads have good mechanical stability and chemical stability, and long preservation time, and the effect is not affected when the fungus is used within 12 months; the luminous fungus gel beads can ensure that the number of active strains is larger than the standard required number in unit volume, and meanwhile, the coating structure is beneficial to preventing the pollution of mixed bacteria to thalli under field conditions. The comparison of Vibrio qinghaiensis Q67 specifically adopted in the embodiment of the invention with other luminous bacteria is shown in the following table 3.
TABLE 3 Table 3
Of course, in other embodiments of the invention, it is also possible to employ Photobacterium brightly, if desiredPhotobacterium phosphoreum T3，Or one of the luminous bacillus ATCC11040 or the vibrio freudenreichii is used as a strain of the luminous bacteria to be embedded and fixed, so that the luminous bacteria gel beads formed by the corresponding strains are prepared, and the luminous bacteria gel beads can achieve the technical effects of the invention when applied to different water quality detection under field conditions.
Example 5
The combined acute toxicity test method for field water quality on-site detection provided by the embodiment of the invention is based on the embodiment 4, and the luminous fungus gel beads prepared in the embodiment 2 are used for combined acute toxicity test for field water quality on-site monitoring industrial pollution sources.
The embodiment utilizes the luminous fungus gel beads prepared by the freshwater luminous fungus Q67 freeze-dried powder preparation to react with different pollutants discharged by partial heavy point pollution source discharge enterprises. In order to have an intuitive and comparable index for biotoxicity, phenol of organic substances and HgCl of inorganic substances are selected 2 Phenol and HgCl were studied as biological positive controls 2 And (3) the combined inhibition of the luminescence of the luminous bacteria gel beads is used as a toxicity evaluation reference substance of the detection result of the luminous bacteria. The results of inhibition of luminescence of Vibrio qinghaiensis Q67 by phenol are shown in Table 4 and FIG. 7.
Phenol and HgCl 2 Standard curves for the luminescence inhibition effect on the luminescent bacteria Q67 show phenol and HgCl 2 Functional relationship of toxic effects on luminescent bacteria Q67.
TABLE 4 inhibition of light emission by phenol on Vibrio qinghaiensis Q67
The correlation equation: y=127.97x+5.48r=0.9978 p >0.01
TABLE 5 inhibition of light emission by mercuric chloride on Vibrio qinghaiensis Q67
The correlation equation: y= 738.9x-12.991 r =0.9461 p >0.01
The results of inhibition of luminescence of Vibrio qinghaiensis Q67 by phenol are shown in Table 5 and FIG. 8.
As can be seen from tables 4 and 5, the linear relation between the light inhibition rate of the luminous bacteria and the concentrations of mercury chloride and phenol is good, which shows that the luminous bacteria gel beads prepared from the Q67 freeze-dried bacteria powder are feasible as objective indexes for evaluating the toxicity of the two toxic and harmful substances.
The invention further utilizes the luminous fungus gel beads prepared by the Q67 freeze-dried fungus powder to carry out biological combined toxicity test of pollutants discharged by different industries, the monitoring data are shown in the following table 6 (luminous fungus biotoxicity of polluted wastewater of each industry), and the monitoring data result shows that the concentration of the pollutants discharged by different industries has good correlation with the luminous inhibition rate of luminous fungus. In the 13 industrial waste water tested, the biological combined toxicity of the waste water in the pesticide industry is strongest (synergistic effect exists between different toxic substances), the EC50 concentration of a sample is 0.1087%, the corresponding mercuric chloride concentration is 5.9938mg/L, the toxicity is minimum in the bleaching and dyeing industry (antagonism exists between different toxic substances), the EC50 of the sample is 92.09%, the corresponding mercuric chloride concentration is 0.1195mg/L, the EC50 of other industries is more than 86.85% (additive effect exists between different toxic substances), the corresponding mercuric chloride concentration is more than 0.1332mg/L, and the sample concentration and the luminous inhibition rate of luminous bacteria have good linear relation in a certain sample concentration range.
TABLE 6
Example 6
The combined acute toxicity test method for field water quality on-site detection provided by the embodiment of the invention is based on the embodiment 4, the luminous fungus gel beads prepared by the embodiment 2 are applied to field water quality on-site test, and the combined acute toxicity of inorganic substances such as arsenic, chromium, lead, cadmium and mercury in a water sample and antibiotics organic toxic substances is tested.
The test results show that when arsenic, chromium, lead and cadmium substances in the aqueous solution exceed EC 50 During the process, the luminous fungus gel beads prepared from the Q67 fungus liquid can obviously inhibit (the inhibition rate is more than 50%) poison EC 50 NaAsO respectively 2 1076mg/Ｌ、CrO 3 722mg/Ｌ、PbCl 2 542 mg/Ｌ、CdCl 2 184mg/L and HgCl 2 145mg/L; the acute toxicity sequence detected by the Q67 bacteria is as follows: hg (II) > Cd (II) > Pd (II) > Cs (VI) > As (III).
In the combined toxicity test between heavy metals, the two toxic substances mainly have synergistic effect, and the combined toxicity effect is ordered by cupric sulfate pentahydrate, cadmium chloride, zinc sulfate heptahydrate, cadmium chloride, cupric sulfate pentahydrate, lead nitrate, zinc sulfate heptahydrate, cupric sulfate pentahydrate, lead nitrate and cadmium chloride; but antagonism is provided between zinc sulfate heptahydrate, potassium dichromate, lead nitrate, potassium dichromate, cadmium chloride and potassium dichromate; zinc sulphate heptahydrate/lead nitrate, copper sulphate pentahydrate/potassium dichromate show additive effect.
In the combined toxicity test of toxic substances of heavy metals and antibiotics, potassium dichromate and doxycycline hydrochloride are synergistic, and the rest arsenic, lead, cadmium and mercury all show antagonism and additive effect; in the combined toxicity test of different antibiotics and antibiotics, antagonism is mainly shown, and the antagonism is strongest by the combination of tetracycline hydrochloride and aureomycin hydrochloride.
Example 7
The combined acute toxicity test method for field water quality on-site detection provided by the embodiment of the invention is based on the embodiment 4, and the luminous fungus gel beads prepared by the embodiment 2 are applied to field water quality on-site test and test of combined toxicity effects of 11 pesticides in a water sample.
Toxic chemical pollution is one of global environmental problems, and pesticides are the compounds with the largest use amount, the widest application range and the highest toxicity in toxic chemicals. The results of the dose-effect fit of 5 soluble pesticides to the luminescent fungus gel beads prepared by Vibrio qinghaiensis Q67 are shown in Table 7; the dose-effect fitting result of 6 poorly water-soluble pesticides on the luminous fungus gel beads prepared by the vibrio qinghaiensis Q67 is shown in table 8, the toxicity of the 11 pesticides can be effectively detected, the detection limit is lower, and the application range is wider; the combined toxic effects between pesticides are mainly manifested by additive and synergistic effects.
TABLE 7
TABLE 8
Example 8
The combined acute toxicity test method for field water quality on-site detection provided by the embodiment of the invention is based on the embodiment 4, the luminous fungus gel beads prepared by the embodiment 2 are applied to field water quality on-site test, and the single toxicity effect of chlorophenols in a water sample is tested to be used as a comparison standard for combined toxicity test in other embodiments.
The half luminescence inhibition concentrations of 2-chlorophenol, 2, 4-dichlorophenol and 2,3, 4-trichlorophenol on Q67 luminophore gel beads were determined in this example and the EC50 values were 99.57, 25.19 and 3.42 mg/L, respectively. The single toxicity of 3 chlorophenols to Vibrio qinghaiensis Q67 is shown in Table 9.
TABLE 9
Remarks: (1) an average value; (2) 95% confidence interval.
Example 9
The combined acute toxicity test method for field water quality on-site detection provided by the embodiment of the invention is based on the embodiment 4, and the luminous fungus gel beads prepared by the embodiment 2 are applied to field water quality on-site test to evaluate the combined acute toxicity of a certain river water body and bottom mud.
In the embodiment, the luminous fungus gel beads prepared by Q67 are adopted, and the luminous fungus combined acute toxicity test is carried out on 8 water body and sediment samples of a certain river, so that the data basis for evaluating the river pollution condition is provided. The water body and the sediment sample comprise a plurality of inorganic matters and organic matters toxic substances. The results of the relative luminous intensity test of luminous bacteria of water samples of 8 river sections are shown in Table 10 (unit:%).
Table 10
From four sampling results of eight sampling points, the river has better water quality in winter, does not show toxicity, and shows a certain degree of toxicity until summer. The water quality of different river reach has obvious difference, and the water quality of upstream river reach such as sampling point 6, sampling point 7, sampling point 8 is obviously better than the water quality of downstream river reach. From the average value of the relative luminous intensity of four times of sampling, the total water quality is basically nontoxic at three points of a sampling point 6, a sampling point 7 and a sampling point 8, the rest river reach shows slight toxicity, and the water quality of the sampling point 3 and the sampling point 5 is at zero boundary points of slight toxicity and medium toxicity.
The test results of the relative luminous intensity of the luminous bacteria of the 8 river reach sediment samples are shown in table 11.
TABLE 11
From four sampling results of eight sampling points, the biological toxicity of the river sediment is similar to that of water, the summer sediment presents a certain degree of toxicity, except for the upstream sampling point 7 and the upstream sampling point 8 of the river reach, the biological activity in the sediment is aggravated due to the rise of the air temperature, and certain toxic substances are generated to inhibit the luminescence of luminous bacteria. From the average value of the relative luminous intensity of four sediment samples, each river-section sludge is in a slightly toxic state, which is probably due to toxic effect of sludge adsorption pollutant on luminous bacteria.
The embodiment of the invention provides the combined acute toxicity test method for field water quality on-site detection, which is characterized in that the field and on-site combined toxicity test is carried out by using the improved portable detection instrument, the porous detection plate and the luminous fungus gel beads, and the detection result is basically the same as the result of toxicity test carried out by using luminous fungus liquid in a laboratory, so that the method can replace a large-volume detection instrument in the traditional technology, and uses fresh culture fungus liquid or on-site reconstituted suspension (working fungus liquid) for experimental detection, reduces the types of detection materials, greatly shortens the flow and workload of on-site combined toxicity detection, and can completely avoid the condition that the number of active strains for detection under the field on-site condition does not reach the standard.
The test results of the embodiment prove that the method for carrying out field water quality field test by using the improved instrument, the detection pore plate and the batch fixed luminous strain prepared by the embedding method to prepare the gel pellets has strong technical advantages and economy. The luminous fungus gel beads can be applied to detecting the toxicity of pollutants within 5 minutes and 30 minutes at maximum after the whole luminous fungus gel beads are naturally thawed on a detection site, and are convenient to operate and quick to use. Compared with the traditional direct bacterial liquid detection, the luminous bacterial gel beads can be rapidly and conveniently applied to site in-situ and combined toxicity tests and single tests, and can also be used for qualitative tests for rapidly detecting pollutants in a short time, so that a large number of operations of sample pretreatment and bacterial liquid preparation are omitted. The invention can mix the bacterial liquid and gel solution prepared in advance in the laboratory in equal volume, and drop the mixed liquid into the calcium carbonate solution through the needle-removing needle cylinder, so as to obtain gel pellets of the luminous bacterial gel beads, which are carried on site after freezing and naturally unfreezing, and then directly carry out qualitative test on a sample water sample, rapidly and qualitatively detect the toxicity of the sample, and the higher the toxicity of the sample is, the lower the peak value of the luminosity of the luminous bacterial gel beads is, and the earlier the peak value appears.
The luminous fungus gel beads provided by the invention are used for fixing surplus luminous strains by an embedding method, so that the concentration of active strains in unit volume can be improved, the operation of preparing fungus liquid by sample pretreatment can be omitted during on-site detection, detection can be implemented directly through the immobilized luminous fungus gel beads after thawing, and the existence of pollutants can be rapidly detected by observing the change of the overall luminous intensity of the luminous strains in the gel beads. In addition, the higher the concentration of the strain in the luminous fungus liquid is when the prepared immobilized gel beads are, the higher the luminous intensity of the luminous fungus gel beads is when the luminous fungus gel beads are detected, so that the change of the luminous fungus gel beads is more favorable for being detected and observed; for the luminous fungus gel beads, the detection method and the detection device which are consistent with the traditional luminous fungus liquid can be used for representing the toxicity of pollutants, and the detection results are more accurate, and meet various requirements of detection standards. The luminous fungus gel beads provided by the invention are easy to produce in batches, can be used as portable test materials, and can meet the qualitative detection requirements of low cost, batch and high efficiency for rapidly detecting acute toxicity of water samples in the field.
According to the embodiment of the invention, through the difference of interaction (permeability and penetrability) between different toxic molecules and the luminous fungus gel beads, the combined toxicity effect (antagonism, independence, addition or synergy) between different types of toxic substances can be qualitatively judged in a one-time detection process, a luminous fungus relative luminescence inhibition rate model based on the concentration of toxic substances such as pesticides is not required to be established, and the quantitative prediction of the combined action of various toxic substances (pesticides or other organic substances and inorganic substances such as heavy metals) on the acute combined toxicity of the luminous fungus can be realized through one-time detection, so that compared with the prior art, the method has the advantages of larger breakthrough, simple and convenient operation and expanding the applicable range of the combined toxicity effect detection substances, and can simultaneously detect the combined toxicity effect between different types of organic substances and inorganic substances, and the corresponding toxicity effect and quantitative concentration can be obtained through one-time detection, thereby greatly simplifying the detection steps and saving the operation time and the detection consumable materials; in some embodiments, the detection time and the detection consumable material of the invention are only 1/3 of that of the prior art, so that the invention has better economy and is suitable for large-scale popularization and application.
In summary, the detection method provided by the embodiments of the invention greatly simplifies detection equipment (tools), detection materials (products) and detection operation steps by cooperatively improving and matching the handheld detector, the detection pore plate and the luminous fungus gel beads, greatly improves the adaptability of the detection environment condition and the detection efficiency of the combined toxicity, improves the stability of the detection result, can realize batch, rapid and combined detection under the field condition of water toxicity, and meets the requirements of fixed-point periodic detection, emergency detection and mechanical combined detection of the water toxicity.
In other embodiments of the present invention, the technical effects described in the present invention may be achieved by performing specific selection of other different schemes within the scope of the detection apparatus, structure, steps, components, strain, ratio, process parameters and conditions, and detection objects described in the present invention, so the present invention is not listed one by one.
The above description is only of the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solution of the present invention or modifications to equivalent embodiments using the methods and technical contents disclosed above, without departing from the scope of the technical solution of the present invention. All equivalent changes of the components, proportions and processes according to the invention are covered in the protection scope of the invention.
Claims (10)
1. The combined acute toxicity test method for field water quality on-site detection is characterized by comprising the following steps of:
s1: preparing a portable handheld water quality acute toxicity detector, wherein the detector comprises a shell, a display panel, a bioluminescence photometer and a detection bin, wherein the display panel, the bioluminescence photometer and the detection bin are arranged in the shell, a detection pore plate is arranged in the detection bin, and a plurality of detection holes are formed in the detection pore plate; the volume of each detection hole arranged on the detection hole plate is 200 mu L;
s2: preparing a plurality of luminous fungus gel beads in batches in advance, ensuring that the number of active luminous strains meeting the detection standard requirement in the coating of each luminous fungus gel bead, and freezing and preserving; the volume of the single luminous fungus gel beads is 50-60 mu L, and the number of active luminous strains in the content of the luminous fungus gel beads is not less than 2 multiplied by 10 on average 5 The method comprises the steps of carrying out a first treatment on the surface of the The pore diameter of the coating is 300-500 nm, and the pore diameter of the hydrophilic network structure inside the coating is 500-800 nm;
s3: carrying a portable handheld water quality acute toxicity detector and frozen batch luminous fungus gel beads to a field water quality detection site, and collecting or preparing a water sample to be detected on the site, wherein the water sample to be detected comprises at least two toxic substances with different molecular diameters;
S4: taking out frozen luminous fungus gel beads in batches, naturally thawing at normal temperature, and recovering activity of luminous fungus in the capsule of each luminous fungus gel bead;
s5: respectively injecting a water sample to be detected into a detection pore plate, then putting a luminous fungus gel bead into each detection pore, and then putting the detection pore plate together with the water sample to be detected and the luminous fungus gel beads into a detection bin in a horizontal state;
s6: in 30 minutes, toxic substances with small molecular diameters in a water sample to be detected firstly pass through the coating to reach the inside of the coating, influence the fluorescence of the luminous strain, form the fluorescence change rate in a first time period, and then organic toxic substances with large molecular diameters also reach the inside of the coating to interact with the toxic substances with small molecular diameters which reach in advance to jointly influence the luminous strain to form the fluorescence change rate in a second time period; using a bioluminescence photometer in a portable handheld water quality acute toxicity detector to respectively acquire and record fluorescence change data of luminous strains in each detection hole according to time sequence, and obtaining the luminous inhibition rate of one toxic substance in the object to be detected in each detection hole to the luminous strains in the luminous fungus gel beads and the luminous inhibition rate of the two toxic substances together to the luminous strains in the luminous fungus gel beads after calculation, thereby obtaining combined acute toxicity data after the combined action of the two toxic substances, and the interaction type of the two toxic substances belongs to synergistic action, additive action or antagonistic action; and then respectively calculating to obtain concentration data of two corresponding toxic substances in the target objects to be detected in each detection hole, recording and outputting the concentration data through a display panel, finishing the on-site combined acute toxicity test of water quality rapid detection under the field condition of one batch, and simultaneously finishing the evaluation of the toxic effect type when the two toxic substances are subjected to combined pollution.
2. The method for combined acute toxicity testing for field water quality on-site detection as recited in claim 1, further comprising the steps of:
s7: after a batch of tests are completed, the detection pore plate is taken out from the detection bin, the completely detected water sample and the luminous fungus gel beads are recovered into a specified container, and the detection pore plate is emptied;
s8: and repeating the steps S4-S7 to finish the combined acute toxicity detection of the water samples to be detected in the subsequent batches until the detection of all the water samples to be detected is finished.
3. The method for combined acute toxicity testing for field water quality on-site detection according to claim 1, wherein the small molecular diameter toxic substances include inorganic substances such as chromium, mercury, zinc, copper, lead, cadmium, arsenic heavy metal ions; the toxic substances with large molecular diameters comprise pesticides, antibiotics, chlorophenols, phenols and phthalate esters of organic substances.
4. The method for combined acute toxicity testing for field water quality on-site detection according to claim 1, wherein,
the luminous fungus gel beads in the step S2 are solid spherical gel beads with complete shapes and smooth surfaces, and the gel beads comprise an envelope and contents completely covered by the envelope; the content is gel liquid including luminous bacteria liquid, and the coating is a film formed by rapidly carrying out gel reaction on the contact surface after the gel liquid contacts with film forming reaction solution; the method comprises the steps that a plurality of luminous strains in luminous bacterial liquid are fixed in gel beads by an envelope, and when a target object to be detected passes through the envelope and reaches the inside of the gel beads, the activity of the luminous strains in the envelope is influenced based on the toxicity concentration of the target object to be detected; the luminous fungus gel beads keep complete shape in the whole water sample detection process, and the coating continuously restrains luminous strains in the film.
5. The method for testing the combined acute toxicity of field water quality on-site detection according to claim 4, wherein the luminous strain is: vibrio qinghaiensis Q67Vibrio qinghaiensis sp.nov.Q67Or luminous bacillusPhotobacterium phosphoreum T3；Or Photobacterium brightens ATCC11040, or Vibrio fischeri.
6. The method for testing the combined acute toxicity of the field water quality on site detection according to claim 4, wherein the luminous fungus gel beads are prepared in advance by adopting the following method:
m1, culturing luminous strains in batches in a laboratory environment or redissolving luminous fungus freeze-dried powder to obtain luminous fungus liquid;
m2, preparing gel liquid including luminous bacterial liquid to obtain luminous bacterial gel liquid with proper luminous bacterial strain concentration content;
m3, preparing a film forming reaction solution, namely dripping the luminous strain gel solution into the film forming reaction solution according to the volume range of 50-60 mu L per drop, wherein the film forming reaction solution rapidly forms a coating on the surface of the drop of the luminous strain gel solution, the coating completely coats and fixes the luminous strain gel solution in spherical gel beads, and the number of active luminous strains in the gel beads is not less than 2 multiplied by 10 on average 5 Obtaining an independent luminous fungus gel bead after dripping once;
M4: repeating the step M3, and dripping the luminous strain gel solution into the film forming reaction solution for a plurality of times to obtain a plurality of luminous strain gel beads in sequence; and (3) freezing and storing the whole luminous fungus gel beads prepared in batches at the temperature of-4 ℃ for transportation, and naturally thawing the luminous fungus gel beads on site and recovering the luminous fungus gel beads to normal temperature when the luminous fungus gel beads are used for detecting the acute toxicity of water quality under field conditions.
7. The method for testing the combined acute toxicity of field water quality on-site detection according to claim 6, wherein the step of culturing the luminous strains in batches in M1 is as follows:
m11: preparing a culture medium: preparing the mixture in proportion to have pH of 9 and preparing the mixture from component KH 2 PO 4 、Na 2 HPO 4 ·12H 2 O、MgSO 4 ·7H 2 O、MgCl 2 ·6H 2 O、CaCl 2 、NaHCO 3 A liquid medium consisting of NaCl, yeast extract, tryptone and glycerol, and adding 2wt% agar to the liquid medium to form a solid medium;
m12: culturing in a culture medium: firstly, placing the luminous strain on the inclined plane of a solid culture medium, and culturing for 24 hours at the temperature of 22 ℃ in a biochemical incubator; inoculating to a triangular flask filled with sterile liquid culture medium, placing into a constant temperature shake incubator, shake culturing at 22deg.C under 160 r/min, and taking out;
m13: and detecting by an enzyme-labeled instrument, and obtaining luminous bacterial liquid for standby when the luminosity is stabilized above 1000000 RLU.
8. The method for testing the combined acute toxicity of field water quality on-site detection according to claim 6, wherein the step M2 of preparing the luminous strain gel solution specifically comprises the following steps:
m21: preparing gel base solution, weighing polyvinyl alcohol and sodium alginate by using an analytical balance, placing the gel base solution into a beaker, adding ultrapure water, heating while continuously stirring, heating until the polyvinyl alcohol and the sodium alginate are completely dissolved, taking out, standing, cooling, and placing into a refrigerator refrigerating chamber at 4 ℃ for refrigerating until bubbles disappear to obtain the gel base solution for later use;
m22: preparing luminous bacterial liquid, namely preparing luminous bacterial liquid with proper luminous bacterial strain concentration content prepared in the step M1 or redissolving luminous bacterial freeze-dried powder to prepare luminous bacterial liquid with proper luminous bacterial strain concentration content, adding glucose into the luminous bacterial liquid to enable the concentration of the glucose in the luminous bacterial liquid to reach 5wt%, or adding sodium chloride to enable the concentration of the sodium chloride in the luminous bacterial liquid to reach 0.8wt%, and then preparing gel base liquid with the step M21 according to the following steps of 1:1 to obtain the luminous strain gel liquid with proper concentration and content of luminous strain.
9. The method for testing the combined acute toxicity of field water quality on-site detection according to claim 8, wherein the step M3 of preparing the luminescent bacteria gel beads specifically comprises the following steps:
M31: preparing a film forming reaction solution: preparing a calcium chloride aqueous solution with the concentration of 20-25 mg/mL as a film forming reaction solution;
m32: sucking the luminous strain gel liquid prepared in the step M2 by using a syringe, then slowly dripping the luminous strain gel liquid in the syringe into a film forming reaction solution according to the volume range of 50-60 mu L per drop, and the film forming reaction solution is coagulated by the dripped luminous strainThe surface of the liquid drop of the glue solution rapidly forms a coating, in particular to sodium alginate and calcium chloride rapidly undergo a gel reaction, and the surface of the dropped liquid drop is coagulated into the coating, so that the dropped gel liquid drop forms solid spherical luminous fungus gel beads with complete shape and smooth surface; the gel liquid of luminous strain is completely coated and fixed in spherical gel beads, and the number of active luminous strains in the gel beads is not less than 2×10 5 Obtaining an independent luminous fungus gel bead after dripping once;
m33, dripping the luminous strain gel liquid sucked into the syringe for multiple times according to the same volume, so that each drop of luminous strain gel liquid is slowly dripped into the calcium chloride solution; after the syringe is dripped, the syringe is extracted again, and the operation is repeated in sequence until the gel liquid of the luminous strain sucked into the syringe is dripped completely, so that a plurality of mutually independent luminous strain gel beads are prepared in batches.
10. The method for testing the combined acute toxicity of the field water quality on-site detection according to claim 6, wherein the step M4 is specifically: repeating the step M3, sucking the luminous fungus gel liquid for a plurality of times by using a needle cylinder, slowly dripping the luminous fungus gel liquid into the film forming reaction solution to obtain luminous fungus gel beads, and preparing a plurality of mutually independent luminous fungus gel beads in batches until the luminous strain gel liquid prepared in the step M2 is completely dripped; and (3) integrally placing the plurality of luminous fungus gel beads prepared in batches, the rest film-forming reaction solution and the container in a refrigerator, freezing and preserving at the temperature of-4 ℃ for transportation, and integrally taking out the luminous fungus gel beads under field conditions of a non-laboratory during use, so that each luminous fungus gel bead is naturally melted on site, restored to normal temperature, and taken out singly in sequence for detecting the acute toxicity of the water sample.
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