CN113009098B - Method for detecting environmental toxic substances by combined biological method - Google Patents

Abstract

The invention discloses a novel method for detecting environmental toxic substances by a combined biological method, which comprises the following steps: collecting an environmental sample specimen to be detected, and judging the type and content of toxic substances in the environment of the specimen to be detected according to three test results by respectively performing a seed germination test, a snail killing test and an aquatic micro-animal community killing test. The invention combines the fenugreek and Chinese little greens seeds of two families of plant kingdom with three different phyla animals of animal kingdom, namely, the leptospira of terrestrial Oncorum, the rotifer of aquatic rotifer, the paramecium of ciliate and the water panda of bradycardia, so that the functions of the two families are complementary, and the two families respond to various toxic substances inherent to the earth and synthesized artificially, thereby having ideal broad spectrum and sensitivity. The storage box for detection comprises a box body and a cover body, wherein an inner box I and an inner box II are arranged in the box body and the cover body through folding frames, and fixed disks for fixing test tools are respectively clamped in the inner box I, the inner box II and the box body, so that the storage box is convenient to carry and detect.

Description

Method for detecting environmental toxic substances by combining biological method

Technical Field

The invention belongs to the technical field of environmental toxicity detection, and particularly relates to a novel method for detecting environmental toxic substances by a combined biological method and a storage box thereof.

Background

At present, the detection of centralized water supply, sewage and harmless treatment, water supply for sudden sanitary disasters, terrorism or criminal poison throwing and water supply for military and hygiene in China still takes the physical and chemical conventional indexes as the standard, and can not make a decision on the existence of comprehensive biological toxicity. The current methods for detecting comprehensive toxicity can be divided into: 1. sensory testing method: through the human sense organs such as eyes, nose, mouth, skin and the like, the color, taste and shape are judged according to the experience of the sanitary staff. 2. Biological test method: the toxicity test of large animals is usually carried out on fish (zebrafish), frog, cat and bird (bird nest), and is judged by toxic symptoms or death of the animals. The foreign technology is introduced in China, and two organisms, namely a killing test of aquatic zooplankton Daphnia magna (Daphnia) and a luminous bacteria inhibition test, are used for detection. 3. Plant detection method: the technology for detecting the micronucleus of the broad bean root tip cells is generally recognized, and is a detection method for detecting the chromosomal aberration of plant cells as an index. 4. And (3) chemical detection: chemical analysis methods are methods for detecting phenomena such as precipitation, color change, etc. caused by chemical reactions. According to different reaction carriers, a test paper method and an air detection tube method are produced, qualitative or semi-quantitative detection is realized, the detection level is trace, and the method is suitable for on-site rapid detection. In conventional monitoring, chemical analysis is the most commonly used means, for example, 15 chemical substances checked by the regulations of the sanitary Standard for Drinking Water in China include: fluoride, chloride, arsenic, selenium, mercury, cadmium, chromium (hexavalent), aluminum, silver, nitrate, chloroform, carbon tetrachloride, benzopyrene, DDT, 666, most of the monitoring units are chemical methods, and the latter three items must be separated and measured using gas chromatography. 5. Detecting by an instrument method: flame photometry, which is mainly used for detecting sulfur and phosphorus-containing compounds, such as nerve agents and mustard gas; thin layer chromatography, mainly using drugs or plant toxins for detection; the portable gas chromatography and the portable gas chromatography-mass spectrometry have high sensitivity, accuracy and reliability on the poison detection tool.

The above methods still have disadvantages: 1. all the above tests are aimed at solving the problems of "presence or absence" and "absence or" presence or absence "of a certain toxic substance in a sample (mainly various types of water or water-containing water), and the comprehensive toxicity of the sample water cannot be proved, and further, the problems of" presence or absence of toxic substance "and" inability to use "are answered, and the number of toxic substances and the added toxicity of the toxic substances cannot be predicted. For the standard inspection promulgated by the country for detecting domestic water and sewage, the safety can be ensured theoretically, and in fact, in addition to the common chemical method detection, the chromatography is difficult to develop in the basic level, and the popularization of portable instruments is influenced by high price and easy damage. At first glance, it seems that the method is general in forests, people cannot take measures until the hygienic disaster is reduced, and even if the detection is carried out according to the standard, the possibility of predicting the existence of toxic substances except the detected project cannot be predicted. 2. In the microbial toxicity test, the method for testing the toxicity of the photobacteria is mostly researched at home and abroad to detect the biological toxicity. The bioluminescence photometer of "Microtox" was introduced in 1978 by Beckman, USA, and the toxicity of dirt was detected by using the change of luminescence intensity after the Vibrio fischeri containing specific fluorescent enzyme separated from seawater meets with the poison. In 1985, the scholars in China separated the freshwater type Qinghai luminous vibrio from the inside of the Qinghai lake lunger, and the method is suitable for detecting non-high-salt specimens. Microtox is the most commercially available kit at present and is widely used for detecting environmental biotoxicity. The rapid and accurate determination of the toxicity test of photobacteria is considered to be one of the most approved methods at present. However, this method is a prokaryotic test method, and because of the characteristics of prokaryotes, it is also unavoidable to have defects, such as high resistance of prokaryotic cells to toxic substances as compared with eukaryotic cells, some compounds must be activated by complicated metabolism in eukaryotic animals to exhibit toxicity, and luminescent bacteria lack these metabolic systems. The original living environment of the luminescent bacteria represented by the vibrio fischeri is a natural low-toxicity or harmless environment, is not tolerant to harmful substances of colored and high-concentration salts in a detected standard, is easy to lose activity and even die, and directly influences a detection result. The method for testing the toxicity of the photogenic bacteria is only specified by German environmental department in European Union in the form of legal documents, and the environment detection is carried out in the United states before and after the major festival and events. The popularization of the technology is also influenced by too much specialized fine operation and correction technology and expensive economic cost, and 3. The daphnia magna acute toxicity test is a biological detection method issued by China in 2015 according to international standard ISO6341-1982 for measuring water quality-daphnia magna movement inhibition. In recent years, daphnia magna is mainly used for researching the toxic action of heavy metal ions, organic pollutants and the like in China, and is used for monitoring pollution sources, early warning and monitoring on-line water quality, testing toxic chemicals such as pesticides and partial traditional Chinese medicines, theoretical research is abundant, and summary data of practical application effects are few. The practical application of the daphnia magna is influenced, the breeding technology is too fine, the operation process is too professional, the observation index is too microscopic, no commercialized kit is supplied, and the kit cannot be accepted by the most needed unit, so that the method cannot leave the laboratory of a research unit. In addition, the growth inhibition test of the aquatic algae (chlorella and chlamydomonas) has been proved to be theoretically used for prediction and forecast, and domestic research reports and popularization suggestions are provided, but no action is seen.

To date, biological cells have been recognized as targets for injury by various organic and inorganic substances that contribute to complex biological toxicities. The search for toxicant-sensitive organisms, tissues, single cells and even organelles (e.g., mitochondria, golgi apparatus, etc.) is considered the subject of investigation. The basic approach follows the magnitude of the change of the index of acute and slow toxicity damage before and after the toxicant contacts the experimental organism, and then determines the possibility of the index. It is common to identify complex toxicity test organisms, which are composed of multiple species of organisms such as plants and animals, and multiple life forms such as single cells, tissue blocks and living individuals, as a "bioscreen" or combined detection mechanism called "bio-barrier" for identifying acute, subacute and chronic toxicity tests of many toxic substances, and which are mainly higher-level eukaryotes, which is the reason why medical, pharmaceutical and toxicological studies use higher-level vertebrates, and rarely lower-level animals and plants as models. The use of large animals for acute toxicity detection such as fish for water detection, chicken for gas detection, cat and bird for toxicity detection has been reported, and as a conventional detection, the above methods are original and difficult to quantify and standardize.

Disclosure of Invention

The invention aims to make up for the defects of the prior art, provides a novel method for detecting the comprehensive toxicity of environmental toxic substances by a combined biological method, and mainly solves three types of indicator organisms with high sensitivity, which are not discovered or applied in the professional fields at home and abroad.

In order to achieve the above object, the present invention provides the following technical solutions:

a novel method for detecting environmental toxic substances by combining biological methods comprises the following steps: collecting an environmental sample specimen to be detected, and judging the comprehensive content of toxic substances in the environment of the specimen to be detected according to three test results by respectively performing a seed germination test, a snail killing test and an aquatic micro-animal community killing test.

Further, the environmental sample to be detected is stored in a water sample form, wherein water samples are directly collected due to water pollution, air samples are collected through the portable air absorption tube due to gas pollution, the air samples are detected after absorption of absorption liquid, soil pollution is detected by quantitative soil sampling and water immersion, oscillation, filtration and centrifugal precipitation are carried out in the oscillator, and supernate is collected to serve as the detection samples. Except mainly dealing with the water body sudden health disaster, should consider the detection of air, soil pollution, simulate air and soil pesticide pollution under the experimental condition, prove that the combined biological detection method is practical, but the mark should be pretreated, the absorption tube can be kept in reserve conventionally, including the large-scale U-shaped porous glass plate straw suitable for large-area air pollution, small-scale on-the-spot bubble type absorption tube of movable core that can use, simple in construction, absorption liquid common water, unknown gas pollution can use organic solvent such as acetone, methanol, etc. at the same time, the absorption liquid that obtains is the sample of toxicity detection, organic solvent sample need remove organic solvent before formal test. The contaminated soil is taken out quantitatively, water is added for soaking, an oscillator is placed for oscillation, filtration and centrifugal precipitation are carried out, and supernatant is used for experimental examination. The procedure thereafter is the same as for a body of water.

Further, fenugreek and Chinese little greens seeds are selected as test organisms in the seed germination test. Trigonella foenum graecum belongs to the plant phylum Leguminosae Rong Lu seed. Chinese little greens rapa L.Chinese group is the seed of Brassica plant of Brassicaceae, also called the Shanghai Chinese little greens. The two seeds have great difference in size, but the high germination rate and the fast germination are common points,

furthermore, the seed germination test is to culture the fenugreek seeds and the Chinese little greens seeds in water sample specimens with different dilutions, and judge the biological toxicity of the sample specimens by taking the germination potential and the synthesis of the chlorophyll as observation indexes, wherein the hard skin of the fenugreek seeds is removed by a manual-mechanical method before the germination test, so that the time difference of the fenugreek seeds after the germination of the Chinese little greens is delayed than that of the Chinese little greens is shortened, and the problems that the fenugreek seeds germinate slowly and the Chinese little greens germinate fast and the combination of the two seeds cannot be judged simultaneously are solved.

Further, the snail killing test selects the snail to be tested as the test organism. The Opea gracile belongs to Onychidae of Oplopoda of Onychida of Orchidaceae of Orthosiphon of Orthonchaceae.

Furthermore, the snail killing test is carried out by laying agar in a transparent plastic cup for test, erecting a loquat leaf bar column treated by bacitracin-sucrose, and taking the climbing capacity of the tested snail after eating or fully contacting a water sample specimen as an observation index.

Further, in the aquatic micro-animal community killing test, rotifer, paramecium and giant reed are selected as the test organisms. Rotifer is a multicellular aquatic animal belonging to the phylum Procoeliacea, the class rotifers. Protozoa of the genus sole ciliate of the genus sole cloaca. The water bear is a common name of tardiradade of the family bradycardiaceae, also called as the panda, and is a multicellular organism.

Furthermore, the aquatic micro-animal community killing test is implemented by culturing rotifer, paramecium and giant panda in water sample specimens with different dilutions, using three kinds of hydrobiological microscopes to check 10 fields of vision, taking no live insects as a killing index, and then taking exercise behaviors, insect body structures and function abnormity as reference indexes.

The invention determines the time of three acute toxicity tests: the test is a seed germination potential test of 38h, a killer test of the Brinell snail of 3h and a killer test of aquatic microfauna of 1h, and the determination can be carried out, and the second and third observation determinations can be carried out after the time is respectively prolonged by 48h and 72 h.

The invention also provides a novel storage box of the detection apparatus for detecting the environmental toxic substances by the combined biological method, which comprises a box body, wherein a cover body is rotatably arranged on one side of the box body, a first inner box and a second inner box which are arranged at intervals up and down are sequentially arranged in the box body and the cover body through folding frames, fixed disks for fixing test tools are respectively clamped in the first inner box, the second inner box and the box body, the fixed disks comprise a first fixed disk clamped in the box body, a second fixed disk clamped in the second inner box and a third fixed disk clamped in the first inner box, the first fixed disk is a test bed for the snail killing test, a clamping cavity is arranged at one corner on the first fixed disk, a first vessel for containing a first thin drill snail is clamped in the clamping cavity, clamping holes which are arranged at intervals are also distributed on the first fixed disk, and plastic cups for separate cup feeding are respectively arranged in the clamping holes; the fixed disc II is a test bed for germination experiments, clamping cavities and cavities which are arranged at intervals and used for placing germination seeds and containing aqueous solution are arranged at one corner of the fixed disc II, and containers II containing seeds are clamped in the clamping cavities; the third fixed disk is a frame body for insecticidal experiments, clamping holes which are arranged at intervals and used for clamping test tubes are distributed in the third fixed disk, a fixing cavity for clamping a plastic tube with a cover is arranged in front of the clamping holes, and the plastic tube with the cover is used for storing culture solution of tested aquatic animals.

One side of the cover body is hinged with one side of the box body through a hinge, the other side of the cover body and the other side of the box body are fastened and connected through a buckle, the buckle is installed on the box body, and a clamping groove corresponding to the buckle is formed in the cover body.

The folding leg constitute by the connecting rod, two outsides of the preceding tip of interior box one are connected with two inboards of the preceding tip of box through connecting rod one respectively, and two outsides of the middle part of interior box one are connected with two inboards of the back tip of box through connecting rod two respectively, two outsides of the back tip of interior box one are connected with two outsides of the back tip of interior box two rather than the top through connecting rod three respectively, two outsides of the preceding tip of interior box two are connected with the upper end of connecting rod two is articulated, the rear both sides of interior box one still are articulated respectively through connecting rod four and lid front end inboard.

The connecting rods are characterized in that connecting shafts for connection are respectively arranged at the end parts of the two ends of the first connecting rod, the third connecting rod and the fourth connecting rod and at the two end parts and the middle part of the second connecting rod, tapered ends are respectively arranged at the end parts of the connecting shafts, and a slit for expanding and fixing is arranged in the middle of each tapered end.

The box body, the first inner box and the second inner box are of double-layer structures and respectively comprise an outer shell, and a foam inner box matched with an inner cavity of the outer shell is embedded in the outer shell.

The upper end face of the cover body is provided with a handle.

The upper port of the box body is provided with a step, the cover body is clamped and matched with the step in a limiting mode, limiting holes are distributed in the step, and limiting bulges correspondingly buckled with the limiting holes are distributed in the covering end face of the cover body.

The principle is as follows: this application adopts the structure of a folding receiver for hold new-type joint biological method measuring ring utensil for toxic substance, and through the structure of corresponding test bed or support body in corresponding box body or the box, be used for respectively clamping and place corresponding utensil, satisfy the experimental requirement that the seed germination is experimental, the spiral shell kill experiment is bored to the slim, aquatic primitive biological killing is experimental, if: the fixed disk I in the box body is a test bed for a snail killing test, the clamping cavity and the clamping hole in the fixed disk I meet the requirements of a split charging plastic quilt and a vessel I for containing a thin drill snail killing test, the fixed disk II in the box body I is a test bed II for a germination test, the clamping cavity in the fixed disk II is used for clamping the vessel II containing seeds, the concave cavity is used for containing an aqueous solution and seeds to be detected, the fixed disk III in the box body II is a test tube for an insect killing test and a plastic tube for fixing aquatic articles, the overall structure design is reasonable, the requirements of three tests are met, when the box body, the box body I and the box body II are used, the cover body is opened, the box body I and the box body II can be staggered and unfolded, the requirements of the instruments are convenient to take, the three tests are respectively arranged in corresponding layers, the independence of the tests are guaranteed, the tests are not disordered, the requirements of the tests are met, the cost is low, the carrying is easy, the tests are convenient, various toxic water, soil and the application range is wide.

The invention has the advantages that:

the invention confirms the superiority of the fenugreek seeds and the Chinese little greens seeds in the germination test, creats the mode of peeling the hard skin of the fenugreek seeds in the germination test, shortens the germination time, uses the terrestrial snails to drill finely, uses the agar-loquat leaf column to feed cups for qualitative and quantitative determination of biological toxicity, confirms that the rotifer, the paramecium and the water panda are suitable for being used as biological toxicity detection indicating organisms, and has the potential of popularization, sensitivity and standardization.

The invention is based on theoretical design, and combines the fenugreek and Chinese little greens seeds of two families in the plant kingdom with three animals of different phyla in the animal kingdom, namely, the leptospira tenuis in the terrestrial drilling spirillaceae, the rotifer in the aquatic rotifer, the paramecium in the ciliate and the water-bear in the bradycardia, so as to complement the functions. From taxonomy, five organisms are far away from each other in the biological evolution tree, and the reaction performance is different. "biological sieve" consisting of animals and plants of the two kingdoms five class (family) responds to various toxic substances inherent to the earth and artificially synthesized. From cytological analysis, the response reaction of the toxic matter with killing effect on cell of the other four animal and plant individuals comprising single-cell paramecium and multiple cells is higher than that of one kind of organism or some biological characteristic of organism, such as light emission, biochemical reaction and enzyme system. These five organisms have recognition properties for poisons that threaten their survival, which have been reduced to the extent that the cells (and further organelles) are damaged and die.

The detection method has broad spectrum, can regularly detect the biological (cell) toxicity to toxic and harmful compounds and pesticides no matter single or compound components, and has stable positive correlation between the toxic effect and the concentration. Also shows significant toxicity difference between ordinary sludge and toxic sludge. The method can be used for detecting artificially contaminated soil samples and aerosols, and can obtain biotoxicity results through qualitative and quantitative detection.

The detection method has ideal sensitivity, and the biological toxic substances in one specimen are simultaneously detected through 5 different organisms crossing phyla and striding class (family), so that the risk of missed detection is greatly reduced. In practice, the same poison has no false negative, and no false positive result is found, although the end point titer of the same poison is different from that of 5 organisms. The detection results of various toxic water, soil and air can be clearly judged.

Description of the drawings:

fig. 1 is a schematic structural view of the utility model after being unfolded;

fig. 2 is a schematic structural view after the utility model is folded;

FIG. 3 is a schematic structural diagram of a first fixing disc;

FIG. 4 is a schematic structural view of a second fixing disc;

FIG. 5 is a schematic structural view of a third drawing tray;

reference numerals:

1. a box body; 2. a cover body; 3. a folding frame; 4. an inner box I; 5. an inner box II; 6. a first fixed disc; 7. a second fixed disc; 8. fixing a disc III; 9. a clamping cavity; 10. a first vessel; 11. clamping the hole; 12. a plastic cup; 13. A clamping cavity; 14. a second vessel; 15. a concave cavity; 16. clamping the hole; 17. a fixed cavity; 18. a first connecting rod; 19. A second connecting rod; 20. a third connecting rod; 24. a connecting rod IV; 21. a handle; 22. a step; 23. and (4) clamping the groove.

Detailed Description

The technical scheme of the invention is further explained by combining the specific examples as follows:

1. seed germination test of fenugreek and Chinese little greens

And determining fenugreek seeds and Chinese little greens seeds as selected seeds in the germination test. Repeated mass selection and continuous test observation show that the two plants have the precondition of being the germination test object. The seed source has large market, wide selectable range and easy obtaining of high-purity seeds. The basic conditions of the seeds such as the purity, the water content and the germination rate are guaranteed. The two plants have long medicinal and edible history, high safety and are compatible with people. Dozens of monocotyledon and dicotyledon seeds including the seed of the sand plant haloxylon which is acknowledged to germinate fastest can not meet three conditions for the drawn experiments: (1) the germination time is short, and is preferably within 72 h. (2) The germination rate is preferably 90% or more. (3) The buds can be seen clearly after sprouting, can be measured by hands, are not brittle and tender, and can withstand repeated measurement. The fenugreek is taken as a fast-growing vegetable in vegetable ranks abroad and Chinese little greens at home, and is a variety which provides the fastest fresh vegetable supply market, such as the Chinese little greens, and the period from sowing to harvesting is 18 days fastest.

Experiments prove that the fenugreek and the Chinese little greens seeds can meet the candidate requirements. The germination rate of the fenugreek seeds for medicine is more than 90.0 percent per batch, and the germination rate of the Chinese little greens is 95.0 percent. The germination rates of some vegetable seeds such as amaranthus viridis and potherb mustard are higher, but the white color is about 30 hours later than the fenugreek, the buds are crisp and fine, and the measurement by hands is not easy. The Chinese little greens are exposed to white for 16h, the cotyledons turn green for 36-38h, chlorophyll is generated, the vitality is extremely strong, although the buds are fine, the flexibility is not broken, and the period is at least 48h earlier than that of the green amaranth. The fenugreek seeds are large and the exposure time is 6-8h later than that of the Chinese little greens, but the buds are vigorous and have strong handiness. The germination time of the haloxylon ammodendron is short, green buds can be seen after 5 hours, but the germination rate is less than 5.0%. The germination rate of the pyrethrum seeds purchased on the net is 0. The germination of the green amaranth and the potherb mustard is slow, and the green-turning time needs 91 hours, so the conditions are not met.

Peeled fenugreek seeds were tried for germination testing. In the field sea seed germination test, spinach, caraway, scallion and oat are wrapped by hard skin, 38h germination design is impossible, and the outer skin of fenugreek seeds is also hard. In order to shorten the germination time of the fenugreek seeds, the crusts of the fenugreek seeds are removed by a manual-mechanical method so as to shorten the time difference that the fenugreek seeds germinate later than the Chinese little greens, and under the same culture condition, the germination time of the fenugreek seeds is 8-10 hours earlier than that of the Chinese little greens without peeling, so that the problems that the fenugreek seeds germinate slowly and the Chinese little greens germinate fast, and the two kinds of seeds can not be judged simultaneously when being used together are changed (as shown in table 1).

TABLE 1 Experimental results of 91 hour germination potential of peeled fenugreek and other seeds

Note: and (delta): if <0.1, the seeds are exposed to white, and the bud length cannot be measured.

As can be seen from the results in table 1, the two batches of peeled fenugreek seeds were bared of germs, the germination time of the seeds is advanced, the germination time of the seeds is respectively 0.6 cm and 0.33cm in 22 hours, and the germination time is less than 0.08cm without peeling. Meanwhile, the average value of the bud length of the Chinese little greens seeds is 0.32cm under the same culture condition, which is obviously lower than that of peeled fenugreek buds, the average values of the peeled fenugreek seeds are 0.96 cm and 0.78cm respectively after 38 hours, the peeled fenugreek seeds are not 0.66cm, the average value of the Chinese little greens also reaches 0.66cm, and if the two seeds are used together, the reliability of testing the biological toxicity in a germination test is undoubtedly improved.

The use of germination potential and chlorophyll synthesis in cotyledons as indicators for observation was established. The germination rate and the germination potential of the seeds are two percentages, which are basic indexes for researching the quality of the seeds. In view of the shortest time to find the forecast, it is clear that the rate of germination is not adequate as an index. Theoretically, germination is considered to be the whole process of seed germination from the earliest calculation of seed embryo umbilicus (hiIar) white exposure to the appearance of cotyledons and complete unfolding. The germination capacity of the seeds is researched, the germination rate can reach seven days by using the germination potential calculation generally according to three days. The effect of the toxic substances on fenugreek and Chinese little greens seeds is predicted to obviously lose significance if the germination rate is used. If the germination rate is used as a reference standard, more than 72 days of observation are needed, namely the formation of the Chinese little greens and the Chinese gourd seeds. The germination potential of the Chinese little greens is obviously superior to that of the fenugreek, so the germination potential is used as an index. The regular observation and recording in the seed germination process have the following indexes: respectively recording the exposed white (existing or absent), the bud length (& ltmi & gt), the cotyledon (yellow/green), the stem and the root or the full length (& ltmi & gt) as shown in table 2.

TABLE 2 Observation of the effects of the four chemicals 48h on the growth of fenugreek buds and the cotyledon development of salsola gallinae

1. If the concentration is less than or equal to 0.1CM, the seeds are white and cannot be measured. The amount of exposed white with bud tip is not calculated according to 0.1 CM. 2. White/yellow/green as the ratio of cotyledon color

As seen from the results in Table 2, the cotyledon turning from yellow to green is clear and stable as an indication of the seed development. The peeled fenugreek seeds can be totally yellow and green within 48-38h, which indicates that the seeds can produce chlorophyll. The unpeeled product must be over 72 hours. The herba Solani Lyrati produces chlorophyll earlier than semen Trigonellae cotyledon, and turns green within 38 h. Therefore, the time difference of chlorophyll synthesis is also an available epiphase marker, in addition to using the germination number, bud length, stem height and root length as detailed observation indexes.

Influence of different chemical agents and pesticides on sprouting potential of fenugreek and Chinese little greens seeds and chlorophyll synthesis. The potassium sulfate, potassium chromate, cyhalothrin, phoxim and niclosamide ethanolamine salt are taken as representatives of chemicals and pesticides and are observed for 24h, 36h and 48h respectively, and the results show that all chemical salts including the potassium sulfate which can be used as a chemical fertilizer, pesticides and molluscicides can inhibit white exposure, bud growth and bud elongation at high concentration and kill buds and dissolve sometimes; inhibit synthesis of chlorophyll from cotyledon, and delay greening for 8-10h. Potassium sulfate and phoxim exhibited further bud elongation-promoting effects at low concentrations (see Table 3).

TABLE 3 Experimental results of the effect of four chemicals on sprouting potential of fenugreek and Chinese little greens in 72 hours

Note: the average value of the total length of the germinated seeds is less than or equal to 1.0cm within delta three days is counted as 0.

Meanwhile, when the blank contrast is less than or equal to 1.0, the average of the blank contrasts of other groups can be used as the group average.

When used for detecting the biological toxic substances in the external environment, the method further comprises the following steps:

1. time required for observation: after the experiment begins, in the 24h period, embryo navel white exposure is taken as an index; the bud length of the seeds, the cotyledon length of Huang Bianlu, and the stem height plus the root length are used as independent indexes and recorded in the 36h time period respectively. The placebo pool is a positive reference if the seeds develop normally. If special conditions occur, the control pool or 5 (or 3) grains are dysplastic and cannot be repeated in time, the results of other blank pools of the same batch can be referred to. If only one blank control exists, the row of the experiment pool which is connected with the control pool and has the lowest content can be used as the blank control, the blank control is temporarily replaced to meet the urgent need, and then the test is repeated.

2. Evaluation calculation for influence degree of seed germination potential: the formula: x is the number of ₀ ＝(x ₁ +x ₂ +x ₃ ……+x _n ) And/n. n is assumed to be n samples. x is a radical of a fluorine atom ₀ Are averages.

The mean value represents the bud length, stem height and root length. When the ratio of the length of the assay object to the blank is 25.0% or less, it is considered to be not much different; greater or less than 50.0%, are considered significant; larger and/or smaller than 1.0 or larger than 1, it should be considered very obvious and no complicated mathematical calculations are made. In this case, the Chinese little greens are significantly different from the fenugreek seeds, and the difference between the peeled Chinese little greens and the non-peeled fenugreek seeds is also significant.

3. A blank "conventional" reference value for germination potential should be established in each laboratory. If the blank control pool can not be used as a control due to accidental factors and dysplasia, the blank control pool can be used as a reference according to the time of blank germination tests without samples accumulated in laboratories at ordinary times, and the germination rates and germination potential results obtained in different years, months and days have little difference on the premise of unchanged test conditions. The repeated development appearance within 96h of the fenugreek and the Chinese little greens is regulated as follows:

the 16h fenugreek and Chinese little greens distillate (including extremely short bud) rates are 96.67% and 93.33%, respectively.

The germination (< 0.2 cm) rates of the fenugreek and the Chinese broccoli at 24h are 93.33% and 96.67% respectively.

The 48h fenugreek germination rate (1.5-2.5 cm) is 96.67%, and the Chinese artichoke germination rate (1.0-2.0 cm) is 96.67%.

The germination rate of 96h fenugreek is 90.0%, and the average value of the bud length is 5.61 cm; the germination rate of the Chinese cabbage is 96.67%, and the germination length is 7.20 cm.

As the observation time is prolonged, dead buds appear at the room temperature of more than 30 ℃, and the germination number is reduced to some extent due to autolysis.

When fenugreek and Chinese little greens seeds are used as germination potential to detect environmental toxicants, the following basic conditions are unified:

temperature: indoor natural temperature and incubator temperature are adopted, preferably at 25-30 deg.C, when too low, the germination time of seed is prolonged, and when too high, fenugreek has rotten bud to inhibit root hair growth.

Water for experiment: city tap water, barreled (bottled water), distilled water, deionized water, even clean spring water, well water and river water treated under special conditions can be used according to local conditions.

Making a filter paper bed of appropriate size: the method is a precondition for ensuring the seed germination test to be observed, paper sheets with the size similar to that of a planting pond are cut by using filter paper and facial tissue, the seeds are placed right and evenly, and one drop of paste is dropped on each seed for drying for later use, so that the seeds are prevented from rolling.

And (4) conclusion: the method is used for detecting the environmental biotoxic substances by using peeled (or non-peeled) fenugreek and Chinese little greens seeds to observe the germination vigor and the cotyledon chlorophyll synthesis indexes. The overall reaction trend for salt chemicals (sodium chloride, potassium sulfate, potassium chromate) is to affect whitening, bud elongation at high concentrations, 2-3 times shorter than controls. Along with the reduction of the concentration, the growth stimulation effect is shown, which is 1-2 times of that of the control, the average value of the bud length in the whole culture period is still obviously lower than that of the control, and dead buds appear. The heavy metal Cr ion has far higher damage to bud growth than potassium sulfate and is irreversible. The pesticide phoxim and pyrethrin is used in field spraying and is less than or equal to 1: 300 dilution, has the function of inhibiting germination, and has the function of obviously stimulating growth at low concentration. The cotyledon begins to synthesize chlorophyll from yellow to green, and has obvious marking effect. Synthesizing chlorophyll with the germination potential (white, bud length, root length and fibrous root) as a quantitative index, and preliminarily judging whether the detected specimen has no toxicity or shows the existence of substances stimulating growth although the specimen has no toxicity within 26-28h, so that the direction is pointed out for the next detection. Because the objectivity of germination, root elongation and cotyledon color is strong, and the influence of subjective factors on the quantitative standard is small, the quantitative index is easy to achieve accuracy.

2. Climbing test of leaf strips of Hemicentrotus trifoliatus

The sensitivity of the fine drilling snail to harmful substances in the environment is high, and the fine drilling snail is proved to be sensitive and stable when used for measuring the toxic substances in the external environment and carrying out quantitative detection. The key of the success or failure of the test is that the tested snail is fully and effectively contacted with the tested sample, and the quantitative index can be obtained by eating or contacting with the tested snail to be infected with the virus and further observing the reaction of the infected snail. By utilizing the characteristic that Tian Zuanluo is good for climbing, as a snail climbing tree, the living and dead reactions after contacting with the detected object are displayed by climbing the loquat leaf strips. The tube bottom agar bed has the functions of moisturizing, fixing loquat leaves and bearing test liquid, is very convenient and fast after being repeatedly used, and the experimental results of various test drugs on the thin bur snail in 15 hours are shown in table 4.

TABLE 4 animal 15-hour experimental results using Hemifusus gracilis as index

As can be seen from table 4 above:

non-toxic salts represented by potassium sulfate at 2.0mg × 10 ³ The method is characterized in that Tian Zuanluo in the/L hypertonic solution survives after lh, 3h and 15h, wherein the first pipe raw solution is not moved in place when 3h, and when the first pipe raw solution climbs to the minimum place of 5 cm at 15h, the snail is shown not to die. The hypertonic solution can survive in 7 gradient ranges of 64 times dilution.

Heavy metals such as potassium chromate, with a chromium content of 1.0mg × 10 ³ L displayCytotoxicity is shown. The toxicity of the chromium ions does not show regular death within 1 hour, irregular death occurs within 3 hours, and regular death does not occur until 15 hours. Chromium salts have been shown to have a slow killing effect on snails, which die through "chronic" poisoning.

The organophosphates represented by phoxim have acute toxic effect on Tian Zuanluo. In three periods of exposure to phoxim, the lethal effects stabilized by 3h and all of the third tube volutes died by 15 h. It must be noted that the climbing ability of the 5 th tube of the slim oncomelania which can not climb up after 3 hours is recovered, the tolerance of the slim oncomelania to the adverse factors of the environment is shown to be stronger, the damaged slim oncomelania in the liquid medicine at the concentration is not dead, and the slim oncomelania is revived after 15 hours. Since we found in the following experiments that the killed fine drill snails were washed with clear water repeatedly and observed in a dish, the killed fine drill snails were partially revived without spitting and without visceral protrusion, and were still inserted into the soil after being placed in a soil culture box.

The pyrethroid pesticide has lower killing effect on the snail mites than organic phosphorus. The test snails die at 0.7% dilution concentrations of 1h, 3h and 15h, 1:2 none of the concentrations after dilution were able to kill all of the snails tested.

The niclosamide ethanolamine salt with molluscacidal effect belongs to amide compounds, is a novel ryanodine receptor insecticide and has a good molluscacidal effect.

It should be noted that in order to ensure that the snail and the reagent are fully contacted, after each measurement, the snail is taken down by ophthalmological forceps and put into the liquid medicine of the agar bed at the bottom of the tube, so that the snail climbs again, the dead snail does not move on the agar bed, the live snail climbs upwards and the dead snail climbs to different heights, and the loquat leaf strip is taken out to measure the height (cm) of the snail. The intact snail eats loquat leaf strips, and biting marks of the snail are clearly visible.

Attention is paid to the identification of false death and death of the Hemifusus punctatus. The false death function of the thin drill snail is self-protection reaction, when the danger of unfavorable existence such as vibration, touch, cold and hot, dryness and the like of the external environment occurs, the head part is retracted into the shell and is fixed without moving, but the time is very short. When dry, mucus is secreted to close the shell opening, which is much worse than the protective effect of snail. And (3) testing whether the tested snail dies, and prompting the person to stretch out the head by the temperature of the fingers except for observing whether the climbing ability exists. The two fingers pinch the tail of the screw for a moment, the head and the kiss can be quickly extended out, a pair of yellowish antennae are glittering and translucent, can stretch out and draw back, can inspect the outside, and can continuously expose the footpad without harm. The dead snails do not respond to the temperature of human bodies, such as fine white foams or visceral protrusions in the shells of the snails, which is the characteristic of death of the snails.

The treated loquat leaves are used for feeding pipes, so that the problem that animals cannot leave a laboratory by taking snails as indexes is solved. The vitality of the snail is taken as an index by using the climbing height, so that the experimental operation becomes stable, visual and credible. The determination time of the detection is 3 hours before the detection liquid is added. The feeding tube is developed successfully, the problems of heat preservation, moisture preservation and foodstuff are solved, feeding and tests are skillfully integrated into one tube, and the feeding tube can be opened repeatedly and observed at any time, so that the feeding tube is very convenient. After soaking the snails, the quick-acting pesticide can lead the sensitive snails to lose the climbing capability through lh, the result is more stable after 3h, the preliminary judgment can be made, and certainly, the experiment should be kept for 15h or longer and then the final judgment is made.

3. Aquatic micro-animal community killing test

In the test, the aquatic animal community insect liquid obtained by the culture medium of the coked cotton has relatively constant main biological species, rotifers and paramecium are main members, and the water-bear worms and nematodes are frequently detected at a chance. As the rotifers are afraid of oscillation, microscopic examination after centrifugation shows that the concentrated rotifers almost all die, the water-bearing bombus becomes cysts, and the paramecium increases in number and almost does not die. Therefore, concentration by centrifugation should be avoided as much as possible.

As shown in Table 5, the large amount of the test on the medium of the coked cotton shows that 3.4mg × 10 mg of the salt chemicals ³ the/L sodium chloride solution is inhibited for 30min for all aquatic organisms, but the inhibition index is not increased and returns to the high concentration range when the time is prolonged to 1h to 16h, which indicates that large and small aquatic organisms are not completely killed, and the aquatic organisms are reactivated in the culture solution with a certain concentration after adaptation. 2.0mg × 10 ³ L potassium sulfate, no inhibition was shown. Potassium chromate content of 1.0mg x 10 ³ the/L has inhibitory effect in high concentration, and the concentration shows irregular inhibitory effect, and the micro-animal community is toxic to chromium ionsThe resistance is tolerant. Is generally sensitive to pyrethrin and phoxim, has broad spectrum to kill aquatic micro-animal communities and has thorough killing effect. The effect of the niclosamide glycolate on the micro animal community is poorer than that of the niclosamide glycolate.

TABLE 5 Effect of six chemicals on the killing of cultured aquatic micro-animal communities

Note: the method has the advantages of the average value: mean of number of macroprotozoa/minibodies per field of view of low power lens. All 0's are 10 patients without active organisms.

The average value is less than or equal to 1.0 in terms of 0.

The killing effect of the medicament on aquatic micro-animal community can be measured within 30min to 16h, and is relatively stable in most time periods. The 4 th and 5 th tubes of 0.7% dilution of pyrethrin and phoxim still have strong killing effect. Niclosamide belongs to amides, has stomach toxicity, mainly affects the oxygen consumption of snail tissues and fades away, and the peak period of killing the snails is two days after the administration.

In order to study the death of rotifers and paramecium by contacting salt pesticides in a shorter period of time, the death of micro-animal communities is observed for 10min, 30min and 60min, and the results are shown in table 6.

TABLE 6 Observation of the 1-hour killing effect of paramecium roridum

Note: delta 0/0 represents the caterpillar fungus number/small aquatic micro animal community number of small caterpillar fungus and the like in the microscopic low power visual field.

The sodium chloride and potassium chromate can kill rotifer and paramecium at 10min, enhance at 30min, and recover to original position at 60 min. The phoxim shows strong killing effect from 10min, slightly decreases 30min, and recovers to high dilution range after 60min, and the total killing effect of the niclosamide is obvious, but the fluctuation is large.

And (3) judging the titer of the insecticidal drug by microscopic examination: the key of the test is that the number of the original organisms is counted by each visual field by using a low power microscope (20 multiplied by 10) to deduce the existence and concentration of harmful substances in the detected liquid. The rotifer has various expressions in the detected liquid which is not suitable for survival: death, dying, or turning, some stiffness is still, and only twisting of the tail part can show that the death is not present. The paramecium has more struggling performance in harmful liquid medicine than rotifer, showing crazy swimming, belly turning, revolving and in-situ rotating and rounding. Particularly, after the potassium chromate solution is contacted with the rotifers and paramecium, the polypide struggles strongly, and the resistance of the rotifers is much weaker than that of the paramecium.

Dividing microscopic examination into 'present' and 'absent': the judgment standard for the existence or nonexistence of the microscopic examination result is that the existence means that one of the rotifer and the paramecium is required to exist, and possibly other raw aquatic organisms exist, and the number of the raw aquatic organisms can be counted visually. Or when the number of the insects is large and small, the Newbo counting plate is also used for counting. Rotifer and paramecium with large size are not suitable for counting by this method. For the determination of "none", ten visual fields were examined for no live target worm.

In liquids with high contents of salts, in particular heavy metal salts, the survival of aquatic micro-animal communities is harmed. At this time, the vitality is reduced, and the movement direction is deviated and rigid until the vitality disappears. Some of them seem to be dead and lose their ability to move, but they recover their ability to move after some time of adaptation.

The results of biological experiments using aquatic micro-zoonosis obtained from cotton culture medium as index show that rotifer, paramecium and other organisms are sensitive to salts, organophosphorus pesticides, pyrethrins and amides, and have significant index significance although there are differences in killing and inhibiting concentrations. The test has clear end point and strong intuition, and the operation is very convenient because the number of the insects to be tested under the microscope is used as an index. The result can be preliminarily judged within 1h, and stable killing effect can be obtained within 3h, and the judgment of the final result can be made. And a vegetable seed germination potential test and a chironomia helix climbing test are performed to simultaneously detect a specimen, so that harmful substances to the survival, growth and development of organisms can be detected from different angles, and the direction is pointed out for further reconnaissance and investigation.

Taking a common water sample for submission as an example, the time and place of sampling, the form and environment of the water sample during water sample collection are checked in detail for the submission person and recorded.

A. Water sample specimen treatment: and determining whether pretreatment is needed according to the visual measurement result of a water sample, wherein the most basic requirements are transparency and no visible substances, and a centrifugal sedimentation method, an absorbent cotton method and a filter paper filtration method can be selected. Covered with an oil film and not removed. Many poisons are oily, but do not interfere with further testing, do not affect clarity, and do not harm the seed, the animal being tested, for other purposes than testing.

B. The pH was measured and recorded. The pH range is 6-10. Too high or too low to be corrected with phosphate buffer. No hydrochloric acid or sodium oxide solution is used to adjust the pH.

C. TDS (total soluble solids): the TDS of the detected water sample is measured by using a TDS tester, the TDS is generally not treated within 2000ppm, and the activity and seed germination of the oncomelania gracilis, the rotifer, the paramecium and the water panda are influenced by too high salt content. If the concentration is too high, the test solution can be quantitatively diluted and then used for testing.

D. Fenugreek and Chinese little greens seed germination potential and chlorophyll synthesis ability inhibition test:

the test can be selected from qualitative examination and quantitative examination according to the difference between the requirement and the urgency.

1. And (3) qualitative detection: the aim is to only prove whether toxic substances are contained or not, so the kit is suitable for inspecting a large number of samples, and the negative samples are not further diluted, thereby saving test materials and time.

2. And (3) quantitative detection: for those who are known to contain toxic substances, have positive qualitative inspection, are highly suspected of being contaminated, or have to know the biological toxicity content of the discharged sewage.

3. The difference between qualitative and quantitative is the difference in dilution factor. The qualitative detection can be only provided with a raw water pipe, 1:2 dilution of two tubes, and 3 rd tube as blank control. The quantitative detection can be provided with 1-9 tubes for dilution by multiple times, and the 10 th tube is used as blank control. In order to prevent the occurrence of non-germination due to the difficulty in mastering the seed quality and influence on judgment, a double blank control can be set.

4. The qualitative test operation process comprises the following steps:

(1) Taking 8 small test tubes with the capacity of 5ml, placing a test tube rack, arranging the test tubes in 2 rows, marking stock solution and 1:2, control 1, control 2;

(2) Adding 1.0ml of tap water or bottled water into each tube except the No. 1 tube;

(3) Adding 1ml of detected water into the 1 st tube, adding 1ml into the 2 nd tube, fully mixing with the water sample, taking out 1ml and discarding;

(4) Taking a 5 multiplied by 4 shrinkage cavity plastic plate, wherein the volume of each cavity is 3.0 multiplied by 2.0 multiplied by 1.5 cm;

(5) Cutting paper pieces with the size similar to that of the bottom of the concave hole by using filter paper or facial tissue, lightly paving the bottom of the hole, preparing in advance to prevent the seeds from rolling, clamping 3 (or 5) full tested seeds by using tweezers, uniformly placing the seeds on the filter paper for later use (0.5% agar or 1 drop of 1.0% starch solution can be dripped, and fixing the seeds not to roll after drying);

(6) The finished product of the non-detection box is self-made by a plastic plate with a concave hole, filter paper pieces with the area similar to the bottom area of the concave hole are firstly cut and laid, and the selected fenugreek and Chinese little greens seeds are respectively picked by an ophthalmic forceps, and 3 particles are put in each hole for standby.

(7) And (3) respectively sucking the stock solution and the liquid mixture by using a quantitative capillary dropper, wherein the liquid mixture comprises: 2, 0.5ml of diluted water sample to be detected, wherein the noisy water can be drained without overflowing, and the seeds are wetted to a certain degree;

(8) Covering with plastic wrap to prevent water evaporation, and recording at room temperature of 26-30 deg.C.

5. The operation process of the quantitative test is as follows:

the quantitative determination is similar to the qualitative test procedure, except that the dilution is increased, and the test tubes for dilution are numbered 1-8 as the tested water tubes, and 9-10 as the comparison.

(l) Adding 1.0ml of tap water or bottled water into each of 2-10 tubes except the 1 st tube;

(2) Adding 1.0ml of detected water into each of the 1 st and 2 nd tubes, mixing from the 2 nd tube, sucking out 1.0ml to the 3 rd tube, mixing thoroughly, sucking out 1.0ml to the 4 th tube, performing the same operation to 8 tubes, taking out 1.0ml, discarding, and dividing the concentration into: stock solution, 1: 2. 1: 4. 1:8 …:128;

(3) All the filter papers of the wells tested were kept at the level of the water pool, and if the wells were dry, the appropriate amount of dilution or tap water was added to the wells.

6. And (3) observation and result judgment:

(1) Observing and recording at 16h, 22h and 38h respectively, wherein the movement is gentle, the tweezers do not clamp the embryo, and the bent bud body is not forced to be straightened during measurement;

(2) Observing and measuring contents: visual inspection

The content includes the time of exposing white seed, extending bud, changing cotyledon into green, appearing time of fibrous root, and observing the synthesis capacity of the leafy green toxin, and the method comprises changing white or light yellow cotyledon into green, taking germinated seed by forceps of ophthalmology, measuring the bud length (mm) one by using a small meter ruler and recording;

(3) And (3) determining the biotoxicity:

the ratio is mostly adopted, namely the measured value mean value is divided into the blank control group mean value, the obtained result is explained by being more than or less than 1.0, the method is simple and visual, and the statistical cost and the cost are not needed. The treatment was carried out in the following manner for the difference that needs to be further revealed.

Equation 1:

(assay group mean-blank control mean) ÷ blank control mean = x%.

And (3) judging: not less than 25.0% and mild toxicity.

Greater than or equal to 50.0%, and moderate toxicity.

Greater than or equal to 100.0 percent, which is severe toxicity.

Equation 2:

mean value formula: x is the number of ₀ ＝(x ₁ +x ₂ +x ₃ ……+x _n ) And/n. n is assumed to be n samples, x ₀ Is an average value, also called mean value.

E. Killing test of the fine drill snail:

and (3) carrying out a second test on the same water sample, namely using the oncomelania gracilis as an index animal to detect whether toxic substances of the oncomelania gracilis exist in the water sample.

1. And selecting a sufficient number of adult snails from the earthwork box for culturing the snails to protect the snail eggs and young snails from being buried deeply and dead. Separately feeding the test grown snails into a plastic square box with a cover, laying filter paper below the box, adding water until the water reaches the minimum, and soaking fresh loquat leaves (or fresh branches of Chinese wolfberry with leaves) in a bacitracin-sucrose solution for more than 3 hours for eating.

2. The rigid transparent plastic pipe for the supply test is 5.0 cm in volume, 3.0 cm in radius, provided with the cover and provided with the air holes. Before the test, 2ml of 0.5% agar was added to each tube, and 1 treated loquat leaf strip was inserted. The leaf strips are 5.0 multiplied by 1.0cm, and are stored in an ice box for later use after the agar is solidified.

3. The qualitative and quantitative detection and dilution method and process of the detected water sample are carried out according to the above steps.

4. After the dilution is finished, 1.0ml of diluted water sample with different concentrations is added into each tube. The control tube was bottled water. Taking 2 (or 4) adult chironomus gracilis with tweezers, making the chironomus gracilis fully contact with water sample, recording time, opening the cover at regular time to observe, keeping the dead chironomus gracilis at the bottom of the tube, and allowing the living snails to climb up along the loquat leaves automatically. And measuring the height of climbing (cm) by using a millet ruler, and recording.

5. Two types of poisoning tables exist after the snail is infected with the virus. First, the screw has a white fine foam accumulated, called spitting foam. More importantly, the internal organs protrude from the small-leaved lemmaphyllum and are yellowish-white. Secondly, the infected loquat leaves are in an dying state and lie on the water or slightly climb up the loquat leaf column without climbing any more after being infected with the toxin, and the poisoning is shown when the loquat leaves out of the water.

6. The slim drill screws are sensitive to environmental changes. It can quickly react to the change of pH and osmotic pressure, and if high-salt solution and peracid and alkali solution are added, it can quickly climb up to escape.

7. When encountering unfavorable living environment, the chironomus is good at false death, and the identification needs to be paid attention. The method comprises the following steps: taking snail with tweezers, pinching the tail of snail shell with fingers, observing with light, exposing head and kiss of live snail for one minute, observing a pair of yellowish antennae which are glittering and translucent, stretching and peeping outside, and exposing foot without harm. Dead snails do not respond to human finger temperature. When further observation is needed, the patient can be taken out by using forceps, washed repeatedly by using experimental water, placed in a culture dish, carefully and visually observed, live crawled, dead feces are dead and dead, and the internal organs are gradually dried by . The snail poisoned in the heavy metal and low concentration pesticide solution loses the climbing ability and does not die, and the snail poisoned in the low concentration pesticide solution is drilled into the soil after being washed.

8. In an actual experiment, the true and false death of the snail is difficult to judge in a short time, so that three poisoning expressions of death, dying and temporary loss of the climbing capability are used in the thin snail drilling with the climbing capability ([ cm ]) being inhibited as an index, namely the climbing capability is lost or weakened.

9. Judging time: the leptospira punctatus belongs to small conch, has a complex tissue structure, is more complex in metabolic process after being contacted with poison than rotifer and paramecium, and has longer acute poisoning display time. Three time periods of 1h, 3h and 15h are drawn up for observation, and 3h is considered to be basically capable of determining whether the biotoxicity contained in the sample exists or not. If necessary, the test sample can be observed and recorded in the original test condition for 15h and 24h respectively.

10. And (4) judging a result: the test reveals the inhibition (killing) effect by comparing the mean value of the per-pool snail climbing height (cm) with the blank control mean value or comparing the total mean value of all titers with the blank control mean value. If it is desired to reveal the biological toxicity of the water sample to be tested from another perspective, statistical processing can be performed using the above formula, meaning and interpretation criteria are also applicable.

F. Aquatic micro-animal community killing test:

the test uses three large aquatic animals, namely rotifer, paramecium and water panda, in a microscopic low-power visual field as index animals to qualitatively or quantitatively detect toxic substances in a detected water sample so as to detect whether the water sample has biological toxic substances.

1. The test insect liquid is from the culture liquid of 15 days using xylitol-coked absorbent cotton culture medium. Before the test, 10 visual fields are counted by visual observation, the total number of the insects obtained is divided by 10, and the average number is larger than or equal to 10, so that the insect liquid can be used as the test insect liquid. It is noted that the brownian hemocytometer plate is not suitable for counting. If the culture solution is single or the number of symbiotic insects can not meet the requirement, the concentration can be carried out by a low-speed centrifugation method, so that the insect content is increased. However, the centrifugation method is not suitable for rotifers, and shaking and centrifugation can cause the death of rotifers.

2. The method is characterized in that the fish tank water filtered by goldfish organisms is used for primary culture and the rotifer, paramecium and water panda are used for subculturing, and the composition of the biological community of the culture solution is greatly different after 15 days. The primary culture solution may contain more rotifers and paramecium, and little water-soluble panda. More are paramecium and other tentatively poorly classified spherical or ovoid organisms with active locomotor activity that can be counted with a neb counting plate. If tested in primary culture, the same field of view is divided into two groups, one of the three (sometimes only one or two) worms and the other (perhaps 1-2) small organisms, observed simultaneously but counted separately. The resistance of 3 large organisms was found to be significantly different from that of small organisms in long-term observation. If the subculture solution is used for the test, only one or at most two index polypides appear in the visual field. However, if the animals are instructed to be panda, the culture solution with high insect content can be obtained 30-60 days before the facultative anaerobic culture condition.

3. The dilution method and procedure for qualitative measurement of the sample water are the same as those in the above-described test. In order to save insect liquid, a micro-test tube method is adopted for diluting a water sample, and a stock solution tube and a water sample dilution tube are arranged in the following steps: 2 the total liquid volume of each tube and blank control tube after dilution is 0.2ml.

4. Adding 0.2ml of tested insect liquid into each tube, fully oscillating and uniformly mixing, placing at the temperature of 20-25 ℃ for sampling by a micro capillary pipette at regular time, dripping a glass slide, not adding a cover glass, and observing and counting by using a 10 multiplied by 10 low power lens.

5. Attention points in determination: although the types and concentrations of toxic substances contained in the water sample to be detected are different, the water sample has killing effect on the 3 large aquatic animals and other small organisms, and the judgment method and the principle are the same. In the experiment, the resistance of large organisms is weak, and the resistance of small organisms is weak. It is probably caused by the difference of action mechanisms of toxic substances such as heavy metals, pesticides and the like due to different aquatic species.

6. The judgment standard and the method are as follows: the methods were also judged to be different due to the different biocenosis with the culture broth. Subculturing a hydraulic species is simple, but the sensitivity to toxic substances and the microscopic appearance are different.

(1) The rotifer is very sensitive to various biological toxic substances, the rotifer body is broken under high concentration, and most of rotifer bodies can only see residual surface film samples, but can not see complete body and internal tissues. Under the action of hypertonic salt or heavy metal, the polypide is complete, but the motion is not directional, the polypide turns over, struggles on the spot, and the tail part can be slowly swung. When the body of the rotifer disappears or the body of the rotifer exists but the rotifer must be completely killed during microscopic examination, the rotifer is judged to be dead. The judgment of complete death requires continuous 10 views. On the contrary, 1 living body was found in 1 visual field, and it was judged as survival and recorded.

(2) Paramecium is the most common organism sensitive to various toxicants. The sports toy is active in harmless environment and beautiful in posture. When high concentration of harmful substance is encountered, the cell membrane is broken and completely disappears in visual field. In low-concentration pesticide and heavy metal liquid, abnormal movements such as directional racing, belly turning, rolling and the like can be caused. The judgment standard is that the insect disappears or the insect dies and flagella completely loses the movement ability, no living body exists in 10 visual fields through continuous observation, and the insect dies completely. The presence of 1 living organism was judged to have no killing effect.

(3) The water-borne panda is sensitive to various poisons and has a higher evolution degree than the above two aquatic small animals. When harmful factors are encountered, the slow-paced animals rapidly curl into a barrel in the presence of water, the dorsal lamella are overlapped, and the elastic horny layer between the lamella contracts into the so-called "barrel stage" (Cask phase), i.e. the shape is like a western wine barrel. The bag body in the state of invisibility has high resistance to low cold, high temperature, oxygen deficiency, dryness and high axial firing. Fresh water is the most basic condition for slow-step class revival. Based on the above biological characteristics of the water-borne pandemics, the following problems must be addressed if the pure water-borne pandemics culture is used to test the biological toxicity of the water sample to be tested.

(1) At ordinary times, a sufficient number of positive cultures of the pandemic must be maintained for testing and passaging. Mainly because the water bear worm proliferates slowly and it is difficult to obtain a high amount of culture solution in a short time, 10 pieces/visual field of culture solution must be picked out from many culture flasks for use, and the low content stress precipitate is concentrated so that its content is not lower than this requirement.

(2) The water-borne panda grown in fresh water is sensitive to hypertonic saline solution, the detected water sample is TDS to be detected with high salt content, and the high-permeability sample is properly treated, such as diluted for use, so as to avoid interference with the test.

(3) The reaction of the water panda when encountering poison, namely the reaction of actively crawling and freely stretching, is rapidly changed within about 5 seconds. The capsule is characterized in that a freely movable insect body is seen by opening eyes under a microscope, the insect body rapidly shrinks from standing to side and seems to swing from side to side, and the shape of the capsule is changed into a round drum shape from a strip shape, namely, the capsule is in an oak wine storage barrel (Cask) shape in English literature, and a faint yellow insect body is changed into a brown capsule. The time is extremely short and is short in the short term.

The tube-slide method is not convenient for observing the deformation process of the water panda by naked eyes, so the tube-slide method is used instead. The operation process is as follows:

a slide glass was previously placed on a microscope stage, and water was supplied for the killing test of the bombus spp.

The qualitative/quantitative analysis water sample dilution tube and the blank control tube do not add the Xiong Chongchong liquid.

Sucking the shaken water-borne panda culture solution by a quantitative capillary pipette, and dripping the solution on the left two positions of the slide glass in each quantitative way without covering glass.

Rapidly observing the quantity and activity of two water-bearing bombus under a low power microscope, sucking the diluted water sample by a tube, quantitatively dripping the diluted water sample with the same amount as the water-bearing bombus culture solution, immediately stirring by a toothpick, rapidly observing, and paying attention to the form change.

The time from the sample adding to the capsule changing is recorded one by one, the time required by the highest dilution capable of triggering the capsule changing is subtracted by the blank control time difference (seconds-s, minutes-min), and the result of the water panda killing test on the qualitative and quantitative toxic substances in the water sample to be detected is obtained.

The storage box comprises a box body 1, a cover body 2 is rotatably mounted on one side of the box body 1, a first inner box 4 and a second inner box 5 which are arranged at intervals up and down are sequentially mounted in the box body 1 and the cover body 2 through a folding frame 3, fixing disks for fixing test tools are respectively clamped in the first inner box 4, the second inner box 5 and the box body 1, the fixing disks comprise a first fixing disk 6 clamped in the box body 1, a second fixing disk 7 clamped in the second inner box 5 and a third fixing disk 8 clamped in the first inner box 4, the first fixing disk 6 is a test bed for killing a snail test, a clamping cavity 9 is formed in one corner of the first fixing disk 6, a first vessel 10 for containing a fine drill snail is clamped in the clamping cavity 9, clamping holes 11 which are arranged at intervals are further distributed in the first fixing disk 6, and plastic cups 12 for feeding separately are respectively clamped in the clamping holes 11; the second fixed disk 7 is a test bed for germination experiments, a corner of the second fixed disk 7 is provided with a clamping cavity 13 and cavities 15 which are arranged at intervals and used for placing germination seeds and containing aqueous solution, and a second container 14 for containing seeds is clamped in the clamping cavity 13; the third fixed disk 8 is a frame body for insecticidal experiments, clamping holes 16 which are arranged at intervals and used for clamping test tubes are distributed on the third fixed disk 8, a fixing cavity 17 for clamping a plastic tube with a cover is arranged in front of the clamping holes 16, and the plastic tube with the cover is used for storing the culture solution of the tested aquatic animals.

Further, one side of the cover body 2 is hinged with one side of the box body 1 through a hinge, the other side of the cover body 2 and the other side of the box body 1 are buckled and connected through a buckle, the buckle is installed on the box body, and a clamping groove corresponding to the buckle in matching is formed in the cover body 2.

Furthermore, the folding frame 3 is composed of connecting rods, two outer sides of the front end part of the first inner box 4 are respectively connected with two inner sides of the front end part of the box body 1 through a first connecting rod 18, two outer sides of the middle part of the first inner box 4 are respectively connected with two inner sides of the rear end part of the box body 1 through a second connecting rod 19, two outer sides of the rear end part of the first inner box 4 are respectively connected with two outer sides of the rear end part of the second inner box 5 above the first inner box through a third connecting rod 20, two outer sides of the front end part of the second inner box 5 are hinged with the upper end part of the second connecting rod 19, and two sides of the rear part of the first inner box 4 are respectively hinged with the inner sides of the front end part of the cover body 2 through a fourth connecting rod 24. Connecting shafts for connection are respectively arranged at the end parts of the two ends of the first connecting rod 18, the third connecting rod 20 and the fourth connecting rod 24 and at the two end parts and the middle part of the second connecting rod 19, tapered end heads are respectively arranged at the end parts of the connecting shafts, and a slit for expanding and fixing is arranged in the middle of the tapered end heads.

Further, box 1, interior box 4 and interior box two 5 are bilayer structure, including the shell body respectively, the shell body embedded be equipped with rather than inner chamber complex foam inner box.

Further, a handle 21 is provided on the upper end surface of the lid body 2.

Further, a step 22 is arranged at the upper port of the box body 1, and the cover body 2 is clamped with the step 22 for limiting matching

Furthermore, a clamping groove 23 for clamping a tool is arranged in front of the fixed cavity 17, a box for containing the tool is clamped in the clamping groove 23, and small scissors, ophthalmological forceps, a small meter ruler and a soft plastic straw are placed in the box.

In the above structure: corresponding facilities of a germination experiment, a snail killing experiment and an insect killing experiment are respectively assembled in the three-layer box structure. The lower-layer box-packed plastic experiment bed capable of containing the fine snail culture medium is provided with nine rows of circular concave holes, and the diameter of each concave hole is 3.3 cm and the depth of 5.2 cm. And loading the transparent plastic cups with the diameter of 3.0 cm and the height of 5.0 cm for separate cup feeding. The middle layer box is used for seed germination experiments, an experiment bed with three rows and nine holes pressed by transparent plastics is embedded, and each hole is 5.0X3.0 x 2.0 cm in length, width and depth. The lower box is used for insecticidal experiments. The test tube rack with the length, width and height of 20.0x8.5 x 5.0 cm is provided with 10 holes in each row and 30 holes in total for discharging diluted liquid. A row of three plastic barrel-shaped tubes with covers for preventing the overflow are additionally arranged in front of the test tube rack, and the base is fixed and can be taken out at any time for storing the culture solution of the aquatic animal to be tested. The bucket height (including the lid) is not more than 5.0 cm, and the diameter 4.O cm. Meanwhile, the utility model is provided with small scissors, ophthalmological forceps, a small ruler and a soft plastic suction tube which are stored in a long square plastic box. In order to improve the accuracy of the measuring and reporting result, the automatic measuring and reporting program is designed by classifying and counting the germination rate of the seeds and the aquatic organism community, and the automatic measuring and reporting program can be completed within half a year.

In the present application: the seed germination test is characterized in that fenugreek seeds and Chinese little greens seeds are cultured in water sample specimens with different dilutions, and the biological toxicity of the sample specimens is judged by taking the germination potential and the synthesis of cotyledon chlorophyll as observation indexes, wherein the hard skin of the fenugreek seeds is removed by a manual-mechanical method before the germination test, so that the time difference of the fenugreek seeds after germination compared with the Chinese little greens is shortened, and the problems that the fenugreek seeds germinate slowly and the Chinese little greens germinate quickly, and the two kinds of seeds can not be judged simultaneously when being used together are solved. In the treponema tenuis killing test, the treponema tenuis is selected as a test organism. The oncomelania gracile belongs to the family of small crude shells of the phylum animalis, the class of gastropoda, the order of phomopoda, the family of Rapana rupestris, and the oncomelania killing test is carried out by laying a bottom on agar in a transparent plastic tube for test, erecting a loquat leaf bar column treated by bacitracin-sucrose, and taking the climbing capacity of the tested snail after eating or fully contacting a water sample specimen as an observation index. In the aquatic protozoon killing test, rotifer, paramecium and water panda are selected as test organisms. Rotifer belongs to the phylum Procoeliaceae, the class rotifers, multicellular aquatic animals. Protozoa of the genus sole ciliate of the genus sole cloaca. The water bear is a common name of tardiradade of the family bradycardiaceae, also called as the panda, and is a multicellular organism. The aquatic protozoon killing test is characterized in that rotifers, paramecium and water panda are cultured in water sample specimens with different dilutions, 10 visual fields are examined by three types of aquatic microscopes, no live insect is taken as a killing index, and movement behaviors, insect body structures and function abnormity are taken as reference indexes.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1. A method for detecting environmental toxic substances by combining biological methods, which is characterized by comprising the following steps: collecting environmental sample specimens to be detected, respectively carrying out seed germination test, snail killing test and aquatic micro animal community killing test, judging comprehensive toxicity and content of toxic substances in the environment where the specimens are detected according to the three test results,

the environmental sample samples to be detected are stored in a water sample form, wherein water samples are directly collected by water pollution, air samples are quantitatively collected by gas pollution through a portable air pump, then the air samples are absorbed through absorption liquid by using an absorption tube to obtain a detection sample, soil pollution is subjected to quantitative soil sampling and water soaking, oscillation and filtration by using an oscillator, centrifugal precipitation, and supernatant is collected as the detection sample,

the absorption liquid is selected from water and organic solvent including acetone or methanol,

the seed germination test selects fenugreek seeds or Chinese little greens seeds as a test organism,

the seed germination test comprises the steps of culturing fenugreek seeds or Chinese little greens seeds in a water sample specimen with quantitative multiple dilution, judging the comprehensive biological toxicity of the sample specimen by taking the germination potential and the synthesis of chlorophyll as observation indexes, wherein the hard skin of the fenugreek seeds is removed by a manual-mechanical method before the germination test,

the snail killing test selects the snail to be tested as the organism,

the aquatic micro-animal community killing test selects rotifer, paramecium or water panda as a test organism, the aquatic micro-animal community killing test examines 10 visual fields by three water biological microscopes through culturing the rotifer, paramecium or water panda in water sample specimens with different dilutions, no live insect is taken as a killing index, and movement behavior, insect body structure and function abnormity are taken as reference indexes,

the receiver of detection apparatus who uses in joint biological method detection environment toxic substance's method, including box (1), lid (2), its characterized in that are installed in one side rotation of box (1): the box body (1) and the cover body (2) are internally and sequentially provided with a first inner box (4) and a second inner box (5) which are arranged at intervals up and down through a folding frame (3), the first inner box (4), the second inner box (5) and the box body (1) are internally and respectively clamped with a fixed disk for fixing test tools, the fixed disk comprises a first fixed disk (6) clamped in the box body (1), a second fixed disk (7) clamped in the second inner box (5) and a third fixed disk (8) clamped in the first inner box (4), the first fixed disk (6) is a test bed for a snail killing test, a clamping cavity (9) is arranged at one corner of the first fixed disk (6), a first vessel (10) for containing a first drill snail is clamped in the clamping cavity (9), clamping holes (11) which are arranged at intervals are further distributed in the first fixed disk (6), and plastic cups (12) for separate feeding are respectively clamped in the clamping holes (11); the second fixed disk (7) is a test bed for germination experiments, a corner of the second fixed disk (7) is provided with a clamping cavity (13) and cavities (15) which are arranged at intervals and used for placing germination seeds and containing aqueous solution, and a second container (14) for containing seeds is clamped in the clamping cavity (13); the fixed disc III (8) is a frame body for insecticidal experiments, clamping holes (16) which are arranged at intervals and used for clamping test tubes are distributed in the fixed disc III (8), a fixing cavity (17) for clamping a plastic tube with a cover is arranged in front of the clamping holes (16), and the plastic tube with the cover is used for storing culture solution of tested aquatic animals;

one side of the cover body (2) is hinged with one side of the box body (1) through a hinge, the other side of the cover body (2) is clamped and fastened with the other side of the box body (1) through a buckle, the buckle is installed on the box body, and a clamping groove correspondingly matched with the buckle is formed in the cover body (2);

the folding frame (3) is composed of connecting rods, two outer sides of the front end part of the first inner box (4) are respectively connected with two inner sides of the front end part of the box body (1) through a first connecting rod (18), two outer sides of the middle part of the first inner box (4) are respectively connected with two inner sides of the rear end part of the box body (1) through a second connecting rod (19), two outer sides of the rear end part of the first inner box (4) are respectively connected with two outer sides of the rear end part of the second inner box (5) above the first inner box through a third connecting rod (20), two outer sides of the front end part of the second inner box (5) are hinged with the upper end part of the second connecting rod (19), and two rear sides of the first inner box (4) are respectively hinged with the inner sides of the front end part of the cover body (2) through a fourth connecting rod (24);

connecting shafts for connection are respectively arranged at the end parts of the first connecting rod (18), the third connecting rod (20) and the fourth connecting rod (24) and at the end parts and the middle parts of the second connecting rod (19), conical end heads are respectively arranged at the end parts of the connecting shafts, and a slit for expansion and fixation is arranged in the middle of each conical end head;

the box body (1) or the cover body (2) or the inner box I (4) and the inner box II (5) are of double-layer structures and respectively comprise an outer shell, and a foam inner box matched with the inner cavity of the outer shell is embedded in the outer shell;

a lifting handle (21) is arranged on the upper end surface of the cover body (2);

a step (22) is arranged at the upper port of the box body (1), and the cover body (2) is clamped and limited with the step (22);

a clamping groove (23) for clamping a tool is arranged in front of the fixed cavity (17), a box for containing the tool is clamped in the clamping groove (23), and small scissors, ophthalmological forceps, a small meter ruler or a soft plastic straw are placed in the box.

2. The method for detecting environmental toxic substances by the combined biological method according to claim 1, wherein the snail-killing test is carried out by laying a bottom of agar in a transparent plastic cup for test, erecting a loquat leaf column treated by bacitracin-sucrose, and taking the climbing capacity of the tested snail after eating or fully contacting a water sample as an observation index.

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