An in situ biological exposure and pollutant-environmental DNA collaborative sampling system

Abstract

The present application discloses an in-situ biological exposure and pollutant-environmental DNA collaborative sampling system, which relates to the technical field of ecological environment monitoring. The water reservoir is connected to the water sampling system of the automatic water quality monitoring station, the raw water pool is connected to the water reservoir through the first sample supply pump, the reference pool is connected to the water reservoir through the extraction column and the second sample supply pump in turn, and the environmental DNA sampler is connected to the water reservoir through the third sample supply pump. The device does not need to be deployed in the water body, is not easy to lose, and is convenient for long-term monitoring; the raw water pool and the reference pool are used for in-situ exposure of aquatic organisms in different ecological niches, and the extraction column and the environmental DNA sampler are used for in-situ collection of pollutant time-weighted average concentration analysis samples and environmental DNA time-mixed samples, respectively, which improves the accuracy of toxicity monitoring results, the representativeness, sensitivity and convenience of pollutant and environmental DNA sampling, realizes the collaborative monitoring of chemistry, toxicology and ecology, and provides a complete chain of information from the existence of chemical pollutants to the impact on the ecosystem.

Description

Technical Field

[0001] The present application relates to the technical field of ecological environment monitoring, and in particular to an in-situ biological exposure and pollutant-environmental DNA collaborative sampling system. Background Art

[0002] A key environmental issue facing aquatic ecosystems is the increasing chemical pressures from human activities. Although most of these chemicals are present in low concentrations, they often appear in complex mixtures with transformation products and unknown chemicals, creating compound pollution ecological risks. Traditional monitoring methods rely on targeted analysis of a small number of chemicals, which are increasingly lacking in reference value and have a weak actual connection with ecological effects.

[0003] In situ biological exposure can measure the toxic effects and bioaccumulation of pollutants under real complex pollution stress, which helps to achieve more targeted and environmentally relevant ecological risk assessments. Existing devices usually carry out exposure by directly fixing the organisms in water bodies after cages, making them vulnerable to flooding, leading to problems such as device loss and non-chemical damage to organisms. In addition, the device itself has no control module and relies on installation in upstream clean water bodies as a reference, which cannot solve the problem of differences in water chemical factors. These factors greatly hinder the feasibility of long-term monitoring and affect the accuracy of monitoring results.

[0004] Theoretically, non-targeted screening methods for pollutants can comprehensively analyze all compounds in the water environment. At present, such samples are generally obtained by passive sampling or collecting instantaneous water samples. However, passive sampling is difficult to determine the sampling volume under complex environmental conditions, making quantitative analysis difficult, and instantaneous water samples cannot reflect the changes in pollutant components and their concentrations over a long period of time.

[0005] Water ecological survey is an important means of identifying water ecological problems and is the basis for water ecological assessment, protection and restoration. In recent years, environmental deoxyribonucleic acid (DNA) technology has developed rapidly as a new means of water ecological survey and has been widely used in target species monitoring and biodiversity surveys. At present, environmental DNA is mainly collected instantaneously by suction filtration or artificial pressure filtration. The capture rate of environmental DNA of low-abundance and occasional species is low, and the labor cost is high. Existing environmental DNA passive samplers are easy to lose, and the collection method needs to be improved.

[0006] In general, the above technologies are still in rapid development, and the existing equipment also has many deficiencies, especially the equipment with in-situ collaborative monitoring capabilities is still blank. Therefore, how to further optimize biological exposure, pollutant and environmental DNA sampling technology, and develop chemical, toxicological and ecological collaborative monitoring equipment has become an urgent problem to be solved. Summary of the invention

[0007] The purpose of this application is to provide an in situ biological exposure and pollutant-environmental DNA collaborative sampling system to solve the following problems: 1) The toxicity monitoring results of the in situ biological exposure device are of low accuracy and are easily invaded by floods and cannot be monitored continuously for a long time; 2) The instantaneous water samples collected by pollutants are poorly representative, and passive sampling is difficult to quantify; 3) The environmental DNA sampling method has a low capture rate for environmental DNA of low-abundance and occasional species, and has high labor costs; 4) There is a lack of collaborative monitoring capabilities for chemistry, toxicology and ecology.

[0008] In order to solve the above technical problems, the present application provides an in-situ biological exposure and pollutant-environmental DNA collaborative sampling system, comprising: a water reservoir, a first sample supply pump, a second sample supply pump, a third sample supply pump, an extraction column for active sampling of pollutants, a water filter and an environmental DNA sampler for environmental DNA sampling, a raw water pool for biological exposure, and a reference pool;

[0009] The water inlet of the water reservoir is used to connect with the water sampling system of the automatic water quality monitoring station. The raw water tank is connected to the water outlet of the water reservoir through the first sample supply pump. The reference tank is connected to the water outlet of the water reservoir through the extraction column and the second sample supply pump in sequence. The extraction column is filled with an adsorbent for enriching pollutants. The environmental DNA sampler is connected to the water outlet of the water reservoir through the third sample supply pump. The environmental DNA sampler is placed in the water filter for enriching environmental DNA.

[0010] In a feasible embodiment, a fine filter and a coarse filter are provided in the water reservoir, and a first water outlet and a second water outlet are provided on the top of the water reservoir, a first water outlet pipe connected to the first sample supply pump is inserted into the water reservoir through the first water outlet and connected to the fine filter, a second water outlet pipe connected to the second sample supply pump is connected to the first water outlet pipe, and a connection point between the second water outlet pipe and the first water outlet pipe is located between the first sample supply pump and the fine filter, a third water outlet pipe connected to the third sample supply pump is inserted into the water reservoir through the second water outlet and connected to the coarse filter; wherein, the pore size of the fine filter is smaller than the pore size of the coarse filter.

[0011] In a feasible embodiment, it also includes a drain pipe, and the drain outlet of the water reservoir, the overflow outlet of the water reservoir, the overflow outlet of the raw water tank, the overflow outlet of the reference tank, and the water filter are all connected to the drain pipe. The drain pipe is used to connect to the drainage system of the automatic water quality monitoring station, and a drain valve is provided on the pipeline connecting the drain outlet of the water reservoir and the drain pipe.

[0012] In a feasible embodiment, a solenoid valve is provided on the pipeline connecting the extraction column and the second sample supply pump.

[0013] In a feasible embodiment, heating rods are provided in the raw water pool and the reference pool, lighting lamps are provided on the outer walls of the raw water pool and the reference pool, and automatic feeders are provided on the top covers of the raw water pool and the reference pool.

[0014] In a feasible embodiment, it further includes a control unit, which is respectively connected to the first sample supply pump, the second sample supply pump, the third sample supply pump, the solenoid valve, the heating rod and the lighting lamp.

[0015] In a feasible embodiment, the environmental DNA sampler includes a quick-connect connector, a filter cartridge, a plug, a filter column, a filter membrane and a sealing ring. The quick-connect connector is plugged into the top of the filter cartridge, and the quick-connect connector is connected to the third water outlet pipe through a Teflon tube. A mounting hole is provided at the bottom of the filter cartridge. The filter column includes a hollow cylinder and a mounting seat provided at the bottom of the hollow cylinder. The hollow cylinder is inserted into the cavity of the filter cartridge through the mounting hole, and the mounting seat is plugged with the filter cartridge through the mounting hole. A through hole connected to the hollow cylinder is provided in the axial direction of the mounting seat. The plug is detachably connected to the top of the hollow cylinder. A plurality of through holes are provided on the side wall of the hollow cylinder. The plurality of through holes are spaced around the circumferential direction of the hollow cylinder. The filter membrane is arranged around the outer wall of the hollow cylinder to cover all the through holes. An annular mounting groove is provided on the circumferential surface of the mounting seat. The sealing ring is arranged in the annular mounting groove, and the sealing ring abuts against the side wall of the mounting hole at the bottom of the filter cartridge.

[0016] In a feasible embodiment, the water filter bucket includes a first cylindrical barrel, a truncated cone barrel and a second cylindrical barrel that are integrally formed and connected in sequence, the diameter of the second cylindrical barrel is smaller than the diameter of the first cylindrical barrel, the environmental DNA sampler is located in the first cylindrical barrel, a base is provided at the bottom of the first cylindrical barrel, the bottom of the mounting seat is connected to the base, the base is provided with a first water outlet connected to the through hole, and a second water outlet is provided at the bottom of the second cylindrical barrel.

[0017] In a feasible embodiment, the raw water tank and the reference tank both include a box body and a top cover arranged on the top of the box body, the first side wall of the box body and the bottom surface of the box body are at an obtuse angle, an overflow port is arranged on the top of the second side wall opposite to the first side wall, and a baffle is arranged on the side close to the second side wall to divide the box body into two chambers, a channel connecting the two chambers is formed between the bottom of the baffle and the bottom of the box body, and the baffle is arranged in the box body with an adjustable position to change the height of the channel.

[0018] In a feasible embodiment, it further comprises a bearing bracket, the water reservoir, the extraction column, the raw water tank and the reference tank are all arranged on the bearing bracket, and the bottom of the bearing bracket is provided with universal wheels.

[0019] The present application provides an in-situ biological exposure and pollutant-environmental DNA collaborative sampling system, comprising: a water reservoir, a first sample supply pump, a second sample supply pump, a third sample supply pump, an extraction column for active sampling of pollutants, a water filter and an environmental DNA sampler for environmental DNA sampling, a raw water pool and a reference pool for biological exposure; the water inlet of the water reservoir is used to be connected to the water sampling system of the automatic water quality monitoring station, the raw water pool is connected to the water outlet of the water reservoir through the first sample supply pump, the reference pool is connected to the water outlet of the water reservoir through the extraction column and the second sample supply pump in sequence, the extraction column is filled with an adsorbent for enriching pollutants, the environmental DNA sampler is connected to the water outlet of the water reservoir through the third sample supply pump, and the environmental DNA sampler is placed in the water filter for enriching environmental DNA. The filtered water extracted by the extraction column provides a reference water sample for biological exposure that basically retains the water chemical characteristics of environmental water samples such as pH, hardness, and salinity, which solves the problem of traditional in-situ biological exposure without a control or large differences in water chemical factors between the control and the raw water, and improves the accuracy of toxicity test results; water sample collection is achieved by connecting the water reservoir to the water sampling system of the automatic water quality monitoring station. The device does not need to be deployed in the water body, is not easy to lose, is simple to maintain, and does not cause non-chemical damage to the exposed organisms due to flooding, thereby improving the feasibility of long-term continuous monitoring; in-situ active sampling of pollutants is completed through solid-phase extraction of the extraction column, and when the test organism shows toxic effects, toxicity traceability can be further achieved; the second sample supply pump can be used to control the flow rate when the water sample passes through the extraction column, so as to obtain accurate and quantitative water samples for quantitative analysis of pollutant components and time-weighted average concentrations of pollutants. It can effectively avoid the problem of poor representativeness of instantaneous water samples when collecting pollutants; the third sample supply pump can continuously deliver water samples to the environmental DNA sampler, so that the environmental DNA sampler can collect environmental DNA for a long time, thereby realizing the collection of mixed samples in section time, improving the capture rate of environmental DNA of low-abundance and occasional species. This process does not require human participation, reducing labor costs; the original water pool and the reference pool are used for in situ exposure of aquatic organisms in different ecological niches, the extraction column is used for active sampling of pollutants and the environmental DNA sampler is used for environmental DNA sampling, which can realize the coordinated monitoring of chemistry, toxicology and ecology, and comprehensively provide complete chain information from the existence of chemical pollutants to their impact on the ecosystem, which is of great significance for accurately identifying the ecological risks of complex pollution in the water environment, deeply analyzing the driving mechanism of biodiversity trends, and scientifically supporting precise decision-making in water ecological management. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 A structural diagram of an in-situ biological exposure and pollutant-environmental DNA collaborative sampling system provided in an embodiment of the present application;

[0022] Figure 2 A schematic diagram of an environmental DNA sampler provided in an embodiment of the present application installed in a water filter;

[0023] Figure 3 An exploded schematic diagram of an environmental DNA sampler provided in an embodiment of the present application;

[0024] Figure 4 A schematic diagram of the flow direction of water samples in an environmental DNA sampler when a plug is installed provided in an embodiment of the present application;

[0025] Figure 5 A schematic diagram of the flow direction of a water sample in an environmental DNA sampler when a plug is not installed provided in an embodiment of the present application;

[0026] Figure 6 A structural diagram of an exposure tank (raw water tank and reference tank) provided in an embodiment of the present application;

[0027] Figure 7 A front view of a system provided by an embodiment of the present application installed on a load-bearing bracket;

[0028] Figure 8 A rear view of a system provided by an embodiment of the present application installed on a load-bearing bracket;

[0029] Fig. 9 A left view of a system provided by an embodiment of the present application installed on a load-bearing bracket;

[0030] Fig.10 A right view of a system provided in an embodiment of the present application installed on a supporting bracket.

[0031] The accompanying drawings are marked as follows: 1-water reservoir, 2-first sample supply pump, 3-second sample supply pump, 4-third sample supply pump, 5-extraction column, 6-water filter, 7-environmental DNA sampler, 8-raw water tank, 9-reference tank, 10-water inlet pipe, 11-fine filter, 12-coarse filter, 13-first water outlet pipe, 14-second water outlet pipe, 15-third water outlet pipe, 16-drain pipe, 17-solenoid valve, 18-drain valve, 19-heating rod, 20-Teflon tube, 21-first side wall, 22-third Two side walls, 23-overflow port, 24-baffle, 25-bearing bracket, 26-universal wheel, 27-lighting lamp, 28-control unit, 601-first cylindrical barrel, 602-truncated cone barrel, 603-second cylindrical barrel, 604-base, 701-quick plug connector, 702-filter cartridge, 703-plug, 704-filter column, 705-filter membrane, 706-sealing ring, 7041-hollow cylinder, 70411-through hole, 7042-mounting seat, 70421-through hole. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0033] The core of this application is to provide an in situ biological exposure and pollutant-environmental DNA collaborative sampling system to improve the accuracy of toxicity monitoring results, the representativeness, sensitivity, and convenience of pollutant and environmental DNA sampling, and the feasibility of long-term continuous monitoring using the existing water quality monitoring network, to achieve collaborative monitoring of water environment chemistry, toxicology, and ecology, to provide a complete chain of information from the presence of chemical pollutants to their impact on the ecosystem, to answer questions such as the ecological risks of complex pollution in the watershed and its driving factors and driving processes, so as to support more scientific and effective environmental management decisions and actions.

[0034] In order to enable those skilled in the art to better understand the present application, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0035] Figure 1 A structural diagram of an in situ biological exposure and pollutant-environmental DNA collaborative sampling system provided in an embodiment of the present application, such as Figure 1As shown, an in-situ biological exposure and pollutant-environmental DNA collaborative sampling system comprises: a water reservoir 1, a first sample supply pump 2, a second sample supply pump 3, a third sample supply pump 4, an extraction column 5 for active sampling of pollutants, a water filter 6 and an environmental DNA sampler 7 for environmental DNA sampling, a raw water pool 8 for biological exposure and a reference pool 9; the water inlet of the water reservoir 1 is used to be connected to the water sampling system of the automatic water quality monitoring station, the raw water pool 8 is connected to the water outlet of the water reservoir 1 through the first sample supply pump 2, the reference pool 9 is connected to the water outlet of the water reservoir 1 through the extraction column 5 and the second sample supply pump 3 in sequence, the extraction column 5 is filled with an adsorbent for enriching pollutants, the environmental DNA sampler 7 is connected to the water outlet of the water reservoir 1 through the third sample supply pump 4, and the environmental DNA sampler 7 is placed in the water filter 6 for enriching environmental DNA.

[0036] The water reservoir 1 in the embodiment of the present application is made of stainless steel, and a water inlet and an overflow are provided near the top of the side wall of the water reservoir 1, a drain is provided at the bottom of the water reservoir 1, and a first water outlet and a second water outlet are provided at the top of the water reservoir 1. The water inlet is connected to a water inlet pipe 10, which is used to connect to the water sampling system of the automatic water quality monitoring station. When the automatic water quality monitoring station starts water sampling, the water sample enters the water reservoir 1, and when the water sample fills the water reservoir 1, it is discharged from the overflow port above.

[0037] The embodiment of the present application does not specifically limit the number and type of the first sample supply pumps 2. The first sample supply pumps 2 can be peristaltic pumps. Each first sample supply pump 2 is connected to a raw water tank 8. When multiple first sample supply pumps 2 and raw water tanks 8 are provided, the same test organisms can be placed in each raw water tank 8 as a parallel, or different test organisms can be placed in each raw water tank 8 to study the impact of water quality on different biological groups. The test organisms can be native fish such as rare minnows or model fish such as zebrafish, as well as benthic animals such as shrimps and river clams or planktonic animals such as large Daphnia. Organisms of different ecological niches can also be used to form a microcosm system for exposure.

[0038] The embodiment of the present application does not limit the number of second sample supply pumps 3. Each second sample supply pump 3 is connected to an extraction column 5 and a reference cell 9 in sequence. The reference cell 9 serves as a control for the raw water cell 8. The number of the reference cells 9 is usually the same as that of the raw water cell 8. After the corresponding test organisms are put into the reference cells, the toxic effects of the composite pollutants on the test organisms are compared and investigated. The second sample supply pump 3 is usually a plunger pump, which can achieve precise control of the flow rate under a high-pressure environment when the water sample passes through the extraction column 5, so as to obtain accurate and quantitative samples for analysis of pollution components and time-weighted average concentrations of pollutants.

[0039] The embodiment of the present application does not specifically limit the number and type of the third sample supply pump 4. The third sample supply pump 4 can be a peristaltic pump. Usually, a third sample supply pump 4 and an environmental DNA sampler 7 are provided for sampling environmental DNA. The water filter 6 can be made of polyvinyl chloride (PVC), and the environmental DNA sampler 7 can be made of copper to prevent bacterial growth.

[0040] Based on the above embodiment, a fine filter 11 and a coarse filter 12 are provided in the water reservoir 1 of the embodiment of the present application, a first water outlet and a second water outlet are provided on the top of the water reservoir 1, a first water outlet pipe 13 connected to the first sample supply pump 2 is inserted into the water reservoir 1 through the first water outlet and connected to the fine filter 11, a second water outlet pipe 14 connected to the second sample supply pump 3 is connected to the first water outlet pipe 13, and the connection point between the second water outlet pipe 14 and the first water outlet pipe 13 is located between the first sample supply pump 2 and the fine filter 11, a third water outlet pipe 15 connected to the third sample supply pump 4 is inserted into the water reservoir 1 through the second water outlet and connected to the coarse filter 12; wherein the aperture of the fine filter 11 is smaller than the aperture of the coarse filter 12. A solenoid valve 17 is provided on the pipeline connecting the extraction column 5 and the second sample supply pump 3.

[0041] like Figure 1 As shown, one end of the first water outlet pipe 13 is connected to the fine filter 11, and the other end of the first water outlet pipe 13 is connected to the first sample supply pump 2 and the raw water pool 8 in sequence; one end of the second water outlet pipe 14 is connected to the fine filter 11 through the first water outlet pipe 13, and the other end of the second water outlet pipe 14 is connected to the second sample supply pump 3, the solenoid valve 17, the extraction column 5 and the reference pool 9 in sequence; one end of the third water outlet pipe 15 is connected to the coarse filter 12, and the other end of the third water outlet pipe 15 is connected to the third sample supply pump 4 and the environmental DNA sampler 7 in sequence. In the embodiment of the present application, the aperture of the fine filter 11 can be 200 meshes. In addition to filtering out leaves and sand, it is also used to further filter out large particles such as mud and sand. By setting the fine filter 11 to filter the water sample before inputting it into the raw water pool 8 and the reference pool 9, the fine filter 11 can reduce the turbidity of the water sample to avoid affecting the accuracy of the biological toxicity monitoring results due to excessive turbidity of the water sample. The pore size of the coarse filter 12 in the embodiment of the present application can be 10 meshes, which is used to filter out impurities such as leaves, sand and stones in the water sample. By setting the coarse filter 12, pipeline blockage is avoided, which facilitates the enrichment of environmental DNA by the environmental DNA sampler 7. Figure 1As shown, the water sample is divided into 4 paths after passing through the fine filter 11, 2 paths of water samples directly enter the raw water pool 8, 2 paths of water samples enter the reference pool 9 after adsorption treatment by the extraction column 5, and the other path of water sample enters the environmental DNA sampler 7 after being filtered by the coarse filter 12. The flow rate of the sample supply pump is adjustable from 5 ml / min to 200 ml / min. The raw water pool 8 is connected to the first water outlet pipe 13 through the Teflon tube 20, the reference pool 9 is connected to the extraction column 5 through the Teflon tube 20, and the environmental DNA sampler 7 is connected to the third water outlet pipe 15 through the Teflon tube 20.

[0042] Based on the above embodiment, the embodiment of the present application further includes a drain pipe 16, the drain outlet of the water reservoir 1, the overflow outlet of the water reservoir 1, the overflow outlet of the raw water tank 8, the water filter 6, and the overflow outlet of the reference tank 9 are all connected to the drain pipe 16, the drain pipe 16 is used to connect the drainage system of the automatic water quality monitoring station, and an emptying valve 18 is provided on the pipeline connecting the drain outlet of the water reservoir 1 and the drain pipe 16. The embodiment of the present application provides the drain pipe 16, and the drain pipe 16 is connected to the drainage system of the automatic water quality monitoring station, so that the drainage of the water reservoir 1, the raw water tank 8, the water filter 6, and the reference tank 9 can be collected and discharged through the drainage system of the automatic monitoring station.

[0043] Based on the above embodiments, the raw water pool 8 and the reference pool 9 of the present application embodiment are both provided with a heating rod 19, the outer walls of the raw water pool 8 and the reference pool 9 are both provided with a lighting lamp 27, and the top covers of the raw water pool 8 and the reference pool 9 are both provided with an automatic feeder. In the present application embodiment, each raw water pool 8 and each reference pool 9 are equipped with a heating rod 19, a lighting lamp 27 and an automatic feeder, and the water temperature, light cycle and light intensity, feeding cycle and feeding amount of the raw water pool 8 and the reference pool 9 can be uniformly controlled according to the habits of the test organisms and the requirements of the toxicity test conditions, thereby eliminating the problem of differences in environmental conditions between traditional in situ biological exposure and control, further improving the accuracy of the monitoring results, and also meeting the needs of long-term continuous exposure of various organisms.

[0044] Based on the above embodiments, the embodiment of the present application also includes a control unit 28, and the control unit 28 is respectively connected to the first sample supply pump 2, the second sample supply pump 3, the third sample supply pump 4, the solenoid valve 17, the heating rod 19 and the lighting lamp 27. Specifically, the control unit 28 includes 2 air circuit breakers, 1 dual-way time relay and 1 programmable logic controller (PLC). An air circuit breaker is used for power control of the heating rod 19 and the lighting lamp 27. The heating rod 19 has a temperature control function, and one time relay is used to control the lighting time of the lighting lamp 27. The water sampling system of the automatic water quality monitoring station is connected to the water pipeline, that is, the water inlet pipe 10 to the water reservoir 1. The water reservoir 1 has a full-load overflow function. When the water reservoir 1 is full, the excess water sample will overflow automatically. Another air circuit breaker and time relay are used to control the sample supply. When the time is up, the first sample supply pump 2 and the third sample supply pump 4 start to supply the sample, and the water sample is supplied to the raw water pool 8 and the environmental DNA sampler 7; the second sample supply pump 3 and the solenoid valve 17 cooperate with the PLC built-in workflow program to control the second sample supply pump 3 (plunger pump) to supply the sample to the reference pool 9 at a constant flow rate after passing through the extraction column 5. The time relay delay time can be set according to the sampling cycle of the water quality automatic monitoring station water sampling system and the flow rate of the sample supply pump. Automatic control is achieved through the control unit 28, which can save labor costs.

[0045] Based on the above embodiments, Figure 2 This is a schematic diagram of an environmental DNA sampler provided in an embodiment of the present application installed in a water filter. Figure 3 This is a schematic diagram of an environmental DNA sampler provided in an embodiment of the present application. Figure 4 A schematic diagram of the flow direction of water samples in an environmental DNA sampler when a plug is installed provided in an embodiment of the present application. Figure 5 A schematic diagram of the flow direction of water samples in an environmental DNA sampler when a plug is not installed is provided in an embodiment of the present application, such as Figures 2 to 5As shown, the environmental DNA sampler 7 in the embodiment of the present application includes a quick-plug connector 701, a filter cartridge 702, a plug 703, a filter column 704, a filter membrane 705 and a sealing ring 706. The quick-plug connector 701 is plugged into the top of the filter cartridge 702, and the quick-plug connector 701 is connected to the third water outlet pipe 15 through the Teflon tube 20. The bottom of the filter cartridge 702 is provided with a mounting hole. The filter column 704 includes a hollow cylinder 7041 and a mounting seat 7042 provided at the bottom of the hollow cylinder 7041. The hollow cylinder 7041 is inserted into the cavity of the filter cartridge 702 through the mounting hole, and the mounting seat 7042 is plugged into the filter cartridge 702 through the mounting hole. The installation The seat 7042 is provided with a through hole 70421 in the axial direction which is connected to the hollow cylinder 7041. The plug 703 is detachably connected to the top of the hollow cylinder 7041. The side wall of the hollow cylinder 7041 is provided with a plurality of through holes 70411. The plurality of through holes 70411 are arranged at intervals in the circumferential direction of the hollow cylinder 7041. The filter membrane 705 is arranged around the outer wall of the hollow cylinder 7041 to cover all the through holes 70411. The circumferential surface of the mounting seat 7042 is provided with an annular mounting groove. The sealing ring 706 is arranged in the annular mounting groove, and the sealing ring 706 abuts against the side wall of the mounting hole at the bottom of the filter cartridge 702.

[0046] The quick-insert connector 701 in the embodiment of the present application can be connected to the top opening of the filter cartridge 702 by threading, and the Teflon tube 20 is connected to the quick-insert connector 701 for conveying water samples into the filter cartridge 702. The filter cartridge 702 is cylindrical with a cavity in the middle, and a mounting hole is provided at the bottom of the filter cartridge 702. The mounting seat 7042 of the filter column 704 is inserted into the mounting hole of the filter cartridge 702, and the sealing ring 706 arranged around the circumference of the mounting seat 7042 abuts against the side wall of the mounting hole to ensure the sealing performance and prevent the water sample in the filter cartridge 702 from flowing out through the gap between the side wall of the mounting seat 7042 and the side wall of the mounting hole. Among them, the hollow cylinder 7041 of the filter column 704 is connected with the mounting seat 7042 in an integral molding manner, and the hollow cylinder 7041 is sleeved in the filter cartridge 702, and the filter column 704 and the filter cartridge 702 are detachably connected to facilitate the replacement of the filter membrane 705. The top opening of the hollow cylinder 7041 is detachably connected to the plug 703 through a thread, and the through hole 70411 provided on the side wall of the hollow cylinder 7041 can be an oblong hole, and the filter membrane 705 is coated on the outer wall of the filter column 704, and the upper and lower ends of the filter membrane 705 can be fixed with rubber bands, so that the filter membrane 705 covers the through hole 70411, so that the water sample in the filter cartridge 702 flows into the interior of the hollow cylinder 7041 through the through hole 70411 after being filtered by the filter membrane 705, and then flows out to the water filter bucket 6 through the through hole 70421 of the mounting seat 7042. The fiber filter membrane coated on the filter column 704 can be made of a material with good adsorption capacity for biological tissues, cells, and nucleic acids, such as polyvinylidene fluoride, polystyrene, polycaprolactone, polyurethane, etc.

[0047] Figure 4The middle plug 703 is installed on the top of the hollow cylinder 7041. The water sample flows into the cavity between the filter cartridge 702 and the filter column 704 through the quick-insert connector 701, and flows into the hollow cylinder 7041 through the filter membrane 705 and the through hole 70411. The water sample in the hollow cylinder 7041 flows into the water filter bucket 6 through the through hole 70421 of the mounting seat 7042, thereby realizing short-term active sampling of environmental DNA by filtration. Figure 5 The middle plug 703 is not installed on the top of the hollow cylinder 7041. It should be noted that the top opening diameter of the filter cartridge 702 used to connect the quick-plug connector 701 is larger than the top opening diameter of the hollow cylinder 7041 used to connect the plug 703, so that most of the water sample flows from the quick-plug connector 701 into the hollow cylinder 7041 of the filter column 704, and a small part of the water sample flows into the cavity of the filter cartridge 702. The water sample flowing into the hollow cylinder 7041 directly flows into the water filter bucket 6 through the through hole 70421 of the mounting seat 7042 without being filtered by the filter membrane 705. The water sample flowing into the filter cartridge 702 flows into the hollow cylinder 7041 through the filter membrane 705 and the through hole 70411, and then flows into the water filter bucket 6 through the through hole 70421 of the mounting seat 7042. When the plug 703 is not installed, the filter membrane 705 is equivalent to being immersed in the water sample, and passive sampling of environmental DNA for a longer time is achieved by adsorption. Since the environmental DNA sampler 7 in the embodiment of the present application is also capable of long-term passive sampling, it is possible to collect mixed samples in cross-section time, thereby improving the capture rate of environmental DNA of low-abundance and occasional species. This process does not require human participation, thereby reducing labor costs. In addition, the environmental DNA sampler 7 in the embodiment of the present application is integrated with the automatic monitoring station and is not directly put into use in the environmental water body, which effectively overcomes the problem of easy loss of the environmental DNA sampler 7.

[0048] Based on the above embodiment, the water filter 6 of the embodiment of the present application includes a first cylindrical barrel 601, a truncated cone barrel 602, and a second cylindrical barrel 603 which are integrally formed and connected in sequence, the diameter of the second cylindrical barrel 603 is smaller than the diameter of the first cylindrical barrel 601, the environmental DNA sampler 7 is located in the first cylindrical barrel 601, the bottom of the first cylindrical barrel 601 is provided with a base 604, the bottom of the mounting seat 7042 is connected to the base 604, the base 604 is provided with a first water outlet connected to the through hole 70421, and the bottom of the second cylindrical barrel 603 is provided with a second water outlet. That is, the water sample of the hollow cylinder 7041 flows out through the through hole 70421 of the mounting seat 7042, the first water outlet of the base 604, and the second water outlet at the bottom of the second cylindrical barrel 603 in sequence, and the second water outlet is connected to the drain pipe 16.

[0049] Based on the above embodiments, Figure 6 A structural diagram of an exposure pool (raw water pool and reference pool) provided in an embodiment of the present application, such as Figure 6As shown, the raw water tank 8 and the reference tank 9 both include a box body and a top cover arranged on the top of the box body, the first side wall 21 of the box body forms an obtuse angle with the bottom surface of the box body, an overflow port 23 is arranged on the top of the second side wall 22 located on the opposite side of the first side wall 21, and a baffle 24 is arranged on the side close to the second side wall 22 to divide the box body into two chambers, a channel connecting the two chambers is formed between the bottom of the baffle 24 and the bottom of the box body, and the baffle 24 is arranged in the box body in an adjustable position to change the height of the channel.

[0050] In the embodiment of the present application, the boxes of the original water pool 8 and the reference pool 9 are both made of inert materials such as polycarbonate (PC), with a volume of 3L and a top cover. The top cover has a water inlet and a feeding hole. A channel connecting the two chambers is left between the bottom of the baffle 24 and the bottom of the box, and the baffle 24 is adjustable in position inside the box to change the height of the channel. The height of the channel can be adjusted according to the size of the test organism to prevent the test organism from escaping. The water inlet at the top of the box can be set close to the first side wall 21. The water sample enters the pool, flows in the direction of the water flow shown in the figure, and flows out from the overflow port 23, so that the metabolic waste and food residues of the test organism can be discharged in time. The embodiment of the present application does not specifically limit how the baffle 24 is adjustably positioned within the box. A plurality of slide grooves may be disposed near the bottom of the side wall of the box. The plurality of slide grooves may be located at the same height of the box, but the widths of the slide grooves are different. The thickness of the baffle 24 gradually decreases from the top to the bottom. Thus, when the baffle 24 is inserted into slide grooves of different widths, the height of the channel formed between the bottom of the baffle 24 and the bottom of the box is different.

[0051] Based on the above embodiments, Figure 7 A front view of a system provided in an embodiment of the present application installed on a supporting bracket, Figure 8 A system provided in an embodiment of the present application is installed on a rear view of a supporting bracket, Fig. 9 A left view of a system provided in an embodiment of the present application installed on a load-bearing bracket, Fig.10 A system provided in an embodiment of the present application is installed on a right side view of a supporting bracket, as shown in FIG. Figures 7 to 10 As shown, the embodiment of the present application further includes a support bracket 25, on which the water reservoir 1, the extraction column 5, the raw water pool 8, and the reference pool 9 are all arranged, and the control unit 28 can also be arranged on the support bracket 25. The embodiment of the present application is provided with universal wheels 26 at the bottom of the support bracket 25 for easy transportation. Figures 7 to 10 As shown, the support bracket 25 includes two layers, the bottom layer is provided with a water reservoir 1 and a reference pool 9, and the top layer is provided with a raw water pool 8 and an extraction column 5. The two raw water pools 8 are located above the water reservoir 1, and the two reference pools 9 are located beside the water reservoir 1, and are all fixed on the bracket by a limiting fixing plate.

[0052] To better understand this application, the following describes the monitoring method and process based on the in situ biological exposure and pollutant-environmental DNA collaborative sampling system, including the following steps:

[0053] (1) System preparation

[0054] According to the type of complex pollution to be monitored and analyzed, an extraction column 5 filled with a broad-spectrum (which can be a combination of multiple adsorbents) or a selective adsorbent is selected to connect the second sample supply pump 3 and the reference cell 9. According to the requirements of active or passive environmental DNA sampling, an environmental DNA sampler 7 with or without a plug 703 is selected to be placed on the base 604 in the water filter 6. The environmental DNA sampler 7 can also be placed or removed during the monitoring process.

[0055] (2) On-site monitoring

[0056] The water sampling system of the automatic water quality monitoring station is turned on, and the water sample enters the water reservoir 1 through the water inlet pipe 10; the first sample supply pump 2 and the third sample supply pump 4 are turned on to pump the water sample into the raw water pool 8 and the environmental DNA sampler 7, and the second sample supply pump 3 and the solenoid valve 17 are turned on. The water sample enters the reference pool 9 after being extracted by the extraction column 5, and active pollutant sampling and environmental DNA sampling begin.

[0057] After setting and balancing environmental conditions such as water temperature, light, water flow and food supply according to the habits of the test organisms and the requirements of the toxicity test, one or several test organisms with different ecological niches are placed in the raw water tank 8 and the reference tank 9 and exposed to the raw water and reference water samples.

[0058] If the biological exposure needs to be carried out for a long time, the extraction column 5 can be replaced during the exposure process to prevent the adsorbent from being saturated with adsorption. The replaced extraction column 5 is brought back to the laboratory and stored below -20°C for analysis.

[0059] (3) Laboratory analysis

[0060] After long-term continuous exposure, the test organisms were sent back to the laboratory and the organisms in the reference pool 9 were used as controls. Based on the selected toxicity endpoints, such as growth, reproduction, tissue pathology, enzyme activity, gene expression and DNA damage, the biological toxicity effects and bioaccumulation status of the complex pollutants in the water body were analyzed, and the ecological risks of the complex pollutants were evaluated using the effect-based trigger value (EBT).

[0061] The extraction column 5 is sent back to the laboratory to elute and analyze the pollutant components in the water environment and their time-weighted average concentrations, and to establish a cross-section water quality fingerprint.

[0062] The filter membrane 705 in the environmental DNA sampler 7 is taken out and sent back to the laboratory to extract DNA, perform polymerase chain reaction (PCR) amplification and sequencing, and analyze the characteristics and trends of target species and biodiversity.

[0063] (4) Mechanism identification

[0064] Mixture toxicity models are used to assess the contribution of each pollutant component to the observed toxic effects through bioequivalent concentration (BEQ), revealing potent chemicals.

[0065] Appropriate models such as the principal response curve (PRC) and threshold-indicating taxonomic analysis (TITAN) were selected to convert ecotoxicity effects into species losses and to quantitatively characterize the damage contribution of complex pollutants to species structural diversity.

[0066] Establish a complete chain of information from pollutants to biological toxicity to ecosystem impacts, and identify the driving mechanisms and processes of trends in aquatic biological structural diversity.

[0067] The in situ biological exposure and pollutant-environmental DNA collaborative sampling system provided in the embodiment of the present application has the following application uses: (1) revealing the toxic effects of complex pollutants in the water environment on aquatic organisms and their potential risks, and providing necessary review and supplementary tools for the existing water environment quality regulatory framework; (2) identifying new pollutants and potent chemicals in the water environment, establishing water quality fingerprints, and providing data support for pollution source analysis and toxic emission reduction; (3) tracking the current status and development trends of aquatic biodiversity and target species, providing indicator data for watershed water ecological assessment and evaluation, and providing scientific support for water ecological protection and restoration; (4) establishing and quantitatively characterizing the complete chain from pollutants to biological toxicity to ecosystem impacts, and providing a basis for priority sorting for management decisions on the watershed water ecological environment.

[0068] The present application aims to provide an integrated system that can eliminate interference from environmental factors and carry out long-term and continuous in situ exposure of organisms in different ecological niches of the water environment and efficient sampling of pollutants and environmental DNA, thereby meeting the needs for coordinated monitoring of watershed chemistry, toxicology and ecology.

[0069] By using the solid phase extraction filtered water that basically retains the water chemical characteristics of environmental water samples such as pH, hardness, and salinity as the reference water sample, the problem of traditional in situ biological exposure without a control or a large difference in water chemical factors between the control and the raw water is solved. By uniformly setting the water temperature, light, water flow, and food supply conditions of the raw water pool and the reference pool, the problem of physical factor differences between the traditional in situ exposure monitoring section and the control section is also solved, thereby greatly improving the accuracy of the monitoring results. At the same time, the equipment of life support systems such as heating rods, lighting, and automatic feeders can meet the long-term in situ exposure needs of organisms in different ecological niches, so it is particularly suitable for monitoring the water quality of rivers and lakes that are currently dominated by chronic effects.

[0070] The in-situ active sampling function of pollutants is realized through solid phase extraction modules such as plunger pumps and extraction columns, which eliminates the need to transport large amounts of water for laboratory analysis of pollutants, greatly improving the sampling efficiency. In particular, based on the actual situation of water environment pollution components and their concentrations changing over time, it solves the problem of time-weighted average concentration sampling of pollutants that cannot be fully achieved by collecting instantaneous water samples.

[0071] By setting up the environmental DNA sampler, you can choose whether to install a plug for active or passive sampling of environmental DNA, which can realize the collection of cross-section environmental DNA time mixed samples, improve the capture rate of low-abundance and occasional species, and reduce sampling costs.

[0072] This application adopts an onshore installation method, and the entire system can be integrated into the existing automatic water quality monitoring station for use. It is of great significance to promote the construction of watershed toxicology and ecology monitoring networks. It also solves the problems of deployment in water that is vulnerable to flooding and causes non-chemical damage to the test organisms, and the easy loss of exposure devices and sampling equipment, thereby improving the operability of long-term monitoring in the field.

[0073] What is particularly critical is that this application can achieve coordinated monitoring of chemistry, toxicology and ecology, which is of great significance for improving the watershed water ecological environment monitoring network, comprehensively providing complete chain information from the existence of chemical pollutants to their impact on the ecosystem, accurately identifying the ecological risks of complex pollution in the water environment, deeply analyzing the driving mechanism of biodiversity trends, and scientifically supporting precise decision-making in water ecological management.

[0074] The above is a detailed introduction to an in situ biological exposure and pollutant-environmental DNA collaborative sampling system provided by the present application. The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same and similar parts between the various embodiments can refer to each other. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

[0075] It should also be noted that, in this specification, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the statement "comprises a ..." does not exclude the presence of other identical elements in the process, method, article or device including the element.

Claims

1. An in situ biological exposure and pollutant-environmental DNA collaborative sampling system, characterized in that: include: A water reservoir (1), a first sample supply pump (2), a second sample supply pump (3), a third sample supply pump (4), an extraction column for active sampling of pollutants (5), a water filter (6) and an environmental DNA sampler (7) for environmental DNA sampling, a raw water pool for biological exposure (8) and a reference pool (9); The water inlet of the water reservoir (1) is used to be connected to the water sampling system of the water quality automatic monitoring station; the raw water pool (8) is connected to the water outlet of the water reservoir (1) via the first sample supply pump (2); the reference pool (9) is connected to the water outlet of the water reservoir (1) via the extraction column (5) and the second sample supply pump (3) in sequence; the extraction column (5) is filled with an adsorbent for enriching pollutants; the environmental DNA sampler (7) is connected to the water outlet of the water reservoir (1) via the third sample supply pump (4); the environmental DNA sampler (7) is placed in the water filter (6) for enriching environmental DNA; The water reservoir (1) is provided with a fine filter (11) and a coarse filter (12). The top of the water reservoir (1) is provided with a first water outlet and a second water outlet. A first water outlet pipe (13) connected to the first sample supply pump (2) is inserted into the water reservoir (1) through the first water outlet and connected to the fine filter (11). A second water outlet pipe (14) connected to the second sample supply pump (3) is connected to the first water outlet pipe (13), and a connection point between the second water outlet pipe (14) and the first water outlet pipe (13) is located between the first sample supply pump (2) and the fine filter (11). A third water outlet pipe (15) connected to the third sample supply pump (4) is inserted into the water reservoir (1) through the second water outlet and connected to the coarse filter (12). The pore size of the fine filter (11) is smaller than the pore size of the coarse filter (12).

2. The in situ biological exposure and pollutant-environmental DNA collaborative sampling system according to claim 1 is characterized in that: It also includes a drainage pipe (16), the drainage outlet of the water reservoir (1), the overflow outlet of the water reservoir (1), the overflow outlet of the raw water tank (8), the overflow outlet of the reference tank (9), and the water filter (6) are all connected to the drainage pipe (16), the drainage pipe (16) is used to connect to the drainage system of the automatic water quality monitoring station, and a drain valve (18) is provided on the pipeline connecting the drainage outlet of the water reservoir (1) and the drainage pipe (16).

3. The in situ biological exposure and pollutant-environmental DNA collaborative sampling system according to claim 2 is characterized in that: A solenoid valve (17) is provided on the pipeline connecting the extraction column (5) and the second sample supply pump (3).

4. The in situ biological exposure and pollutant-environmental DNA collaborative sampling system according to claim 3 is characterized in that: The raw water pool (8) and the reference pool (9) are both provided with heating rods (19), the outer walls of the raw water pool (8) and the reference pool (9) are both provided with lighting lamps (27), and the top covers of the raw water pool (8) and the reference pool (9) are both provided with automatic feeders.

5. The in situ biological exposure and pollutant-environmental DNA collaborative sampling system according to claim 4, characterized in that: It also comprises a control unit (28), wherein the control unit (28) is respectively connected to the first sample supply pump (2), the second sample supply pump (3), the third sample supply pump (4), the solenoid valve (17), the heating rod (19) and the lighting lamp (27).

6. The in situ biological exposure and pollutant-environmental DNA collaborative sampling system according to claim 1, characterized in that: The environmental DNA sampler (7) comprises a quick-insert connector (701), a filter cartridge (702), a plug (703), a filter column (704), a filter membrane (705) and a sealing ring (706), wherein the quick-insert connector (701) is plugged into the top of the filter cartridge (702), the quick-insert connector (701) is connected to the third water outlet pipe (15) via a Teflon tube (20), a mounting hole is provided at the bottom of the filter cartridge (702), the filter column (704) comprises a hollow cylinder (7041) and a mounting seat (7042) provided at the bottom of the hollow cylinder (7041), the hollow cylinder (7041) is inserted into the cavity of the filter cartridge (702) through the mounting hole, and the mounting seat (7042) is plugged into the filter cartridge (702) via the mounting hole, The mounting seat (7042) is provided with a through hole (70421) in the axial direction and connected with the hollow cylinder (7041); the plug (703) is detachably connected to the top of the hollow cylinder (7041); the side wall of the hollow cylinder (7041) is provided with a plurality of through holes (70411); the plurality of through holes (70411) are arranged at intervals in the circumferential direction around the hollow cylinder (7041); the filter membrane (705) is arranged around the outer wall of the hollow cylinder (7041) to cover all the through holes (70411); the circumferential surface of the mounting seat (7042) is provided with an annular mounting groove; the sealing ring (706) is arranged in the annular mounting groove, and the sealing ring (706) abuts against the side wall of the mounting hole at the bottom of the filter cartridge (702).

7. The in situ biological exposure and pollutant-environmental DNA collaborative sampling system according to claim 6, characterized in that: The water filter bucket (6) comprises a first cylindrical barrel (601), a truncated cone barrel (602) and a second cylindrical barrel (603) which are integrally formed and connected in sequence, the diameter of the second cylindrical barrel (603) being smaller than the diameter of the first cylindrical barrel (601), the environmental DNA sampler (7) being located in the first cylindrical barrel (601), the bottom of the first cylindrical barrel (601) being provided with a base (604), the bottom of the mounting seat (7042) being connected to the base (604), the base (604) being provided with a first water outlet which is connected to the through hole (70421), and the bottom of the second cylindrical barrel (603) being provided with a second water outlet.

8. The in situ biological exposure and pollutant-environmental DNA collaborative sampling system according to claim 1, characterized in that: The raw water tank (8) and the reference tank (9) both comprise a box body and a top cover arranged on the top of the box body, the first side wall (21) of the box body and the bottom surface of the box body form an obtuse angle, an overflow port (23) is arranged on the top of the second side wall (22) located on the opposite side of the first side wall (21), a baffle (24) is arranged on the side close to the second side wall (22) to divide the box body into two chambers, a channel connecting the two chambers is formed between the bottom of the baffle (24) and the bottom of the box body, and the baffle (24) is arranged in the box body in an adjustable position to change the height of the channel.

9. The in situ biological exposure and pollutant-environmental DNA collaborative sampling system according to claim 1, characterized in that: It also comprises a bearing bracket (25), on which the water reservoir (1), the extraction column (5), the raw water pool (8) and the reference pool (9) are all arranged, and a universal wheel (26) is provided at the bottom of the bearing bracket (25).

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