WO1995011989A1 - Dosage biologique in vitro concernant la toxicite d'un fluide - Google Patents
Dosage biologique in vitro concernant la toxicite d'un fluide Download PDFInfo
- Publication number
- WO1995011989A1 WO1995011989A1 PCT/US1994/012402 US9412402W WO9511989A1 WO 1995011989 A1 WO1995011989 A1 WO 1995011989A1 US 9412402 W US9412402 W US 9412402W WO 9511989 A1 WO9511989 A1 WO 9511989A1
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- WIPO (PCT)
- Prior art keywords
- toxicant
- fluid
- toxicants
- membrane
- biotargets
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/0004—Gaseous mixtures, e.g. polluted air
- G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
- G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
- G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
- G01N33/0047—Organic compounds
- G01N33/0049—Halogenated organic compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1826—Organic contamination in water
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
Definitions
- This invention relates to the field of testing fluids potentially containing toxicants. More specifically, the invention relates to a device and a method for an in vi tro test which can determine the biological toxicity and toxicity profile of environmental fluid samples such as waste streams, effluents, manufacturing runoffs, sewage streams, air flow, and air and water extracts of environmental samples.
- Toxicants can be present in trace amounts in air, soil, water, or foods and foodstuffs and can produce acute and chronic adverse somatic effects in humans or animals exposed to or ingesting these materials, as well as mutagenic, teratogenic or carcinogenic effects.
- Assays to detect the presence of toxic substances in environmental samples at very low levels and within a reasonable period of time are needed. Ideally, such assays are qualitative as well as quantitative, by indicating the type of chemical substance detected by a positive result. However, at present both qualitative tests to identify an unknown toxicant and accurate quantitative chemical analyses are very slow and expensive processes. Environmental mixtures present special difficulties because one sample may need to be divided into small aliquots to quantitate multiple constituents. Sometimes appropriate analytical methodology and toxicological information is not available to quantitate minute but toxic amounts of individual compounds, particularly new compounds.
- the analyses are further complicated when multiple toxicants are present in environmental samples.
- Bioassays typically measure the response of biological preparations or whole organisms to a sample of unknown constituents but do not identify or quantify the chemicals concerned, except for their biological activity.
- bioassays are based on relatively simple biochemical tests, such as single enzymes or groups of enzymes.
- Another type of bioassay is based on the responses of cells, microorganisms or whole organisms. Whole organism approaches are exemplified by the well known Ames test for mutagenicity which tests the effect of a sample on the mutation rate of a bacterial strain with a known mutation rate and by the Microtox test marketed by Beckman Instruments which assesses the quenching of bioluminescence from Photobacterium phosphoreu as a measure of a substance's toxicity.
- vi tro tests are frequently more sensitive to the basic effects of a chemical and respond at concentrations at least as low as those affecting the whole organism (Ekwall, B., Ann. New York Acad. Sci . (1983) pp. 64-77) . Consequently, appropriate in vi tro systems should help separate the various interacting physical and chemical processes associated with an organism's reaction to injury and enable study of specific actions at the macromolecular level, without the complicating effects of multiple organ systems (Dipple, A., et al., Ann. New York Acad. Sci. (1983) pp. 27-33) . None of the foregoing tests, however, provides a toxicant profile.
- the invention is directed to a method to obtain a qualitative and quantitative toxicant profile of a fluid which method comprises a) contacting said fluid with an assembly of a multiplicity of compositions, each of which compositions reacts with at least one toxicant so as to produce a detectable signal, and wherein said compositions are chosen so that each potential toxicant will react with at least one of said compositions, but no more than one potential toxicant reacts with all of said compositions; b) detecting each of said detectable signals; and c) evaluating said multiplicity of signals to obtain said profile.
- kits comprising at least one film and a plurality of biotargets is provided, the fluid is exposed to the biotargets and at least one change is detected.
- the biotargets and a reagent are combined in a separation device such as a centrifuge tube, to which is added the fluid; toxicants react with the biotargets and produce probes; the fluid is separated and the device is observed for separated probes .
- a separation device such as a centrifuge tube
- toxicants react with the biotargets and produce probes
- the fluid is separated and the device is observed for separated probes .
- nother embodiment employs a kit with a plurality of biotargets, a reagent and a 24-well plate. Standard amounts of biotargets and reagent are placed in each well; fluid is added to at least some of the wells and the wells observed for changes.
- Another embodiment employs a kit with a series of bioassay plates, each of which comprises at least one film, at least one type of biotarget and a housing to hold the film and biotargets.
- Still another embodiment employs a cartridge comprising a series of bioassay plates, each of which has at least one film and at least one type of biotarget, with the biotarget being capable of a change when said biotarget is exposed to a toxicant .
- the cartridge is placed so that the plates contact the waste stream.
- Each plate generates a signal when its biotargets contact toxicants, so that the cartridge generates a profile.
- the profile is used to identify the toxicant or toxicants or type of toxicant .
- Another embodiment employs a biomembrane which is attacked by at least one toxicant; a solution on a first and a second side of the biomembrane such that the first- side solution has a high concentration of electrolytes and the second-side solution has a low concentration of electrolytes; and a means for measuring the potential across the solution on the second side of the membrane.
- the test fluid is added to the solution on the first side of the biomembrane and the electrical potential across the solution on the second side is measured.
- the change in electrical potential from that of a low electrolyte solution indicates a toxicant in the fluid.
- the profiles obtained are useful in identifying a single unknown toxicant as well as a multiplicity of toxicants.
- the plurality of biotargets capable of generating different changes permits profiling the toxicant by the change or changes detected, whereby the toxicant is identified by class or as a particular toxicant.
- Figures 1A through ID are schematic representations of a red blood cell (RBC) (A) , a cutaway view of an RBC (B) , a liposome (C) and a layered biotarget (D) .
- Figure 2A is a schematic representation of a cross- section of a biomembrane, such as an RBC membrane.
- Figure 2A is a schematic representation of a bioequivalent system.
- Figures 4 through 10 are schematic representations of a series of bioassay plates as well as various cartridges for holding the plates .
- Figure 11 is a schematic representation of a pump to move fluid through the cartridge which contains bioassay plates.
- Figure 12 shows a schematic representation of a cross-section of a vial or well containing an assay plate with biotargets.
- Figure 13 is a schematic representation of a cross- section of a vial containing several assay plates.
- Figures 14A and B are schematic representations of assay paddles which are coated with biotarget (s) , biocoatings and optionally film outer coats.
- Figures 15A and B schematically show the effect of a surfactant on a biomembrane separating high- and low- chloride solutions.
- Figure 16 schematically shows a biocoating (top) after it has been in contact with a toxicant (bottom) which breaks up the protein coating.
- Figure 17 is a schematic representation of an effluent alarm system, which can be used to reroute contaminated sewage or levy fines on polluters.
- fluid is meant a liquid or gas.
- a "fluid potentially containing toxicants”, also referred to as a “test fluid” is a fluid to be tested which may contain toxicants. Examples of such test fluids are waste streams from chemical plants, paper processing facilities and other types of manufacturing plants, sewage streams, remediation sites, streams, lakes and oceans. Examples of test gases include airflows in manufacturing plants and other environments.
- Solid samples, such as soil samples, also can be used in the present invention, provided they are first added to a fluid, such as water.
- solid sample extracts can be prepared from such materials as solids, slushes, refinery streams, dump sites, and plant building materials.
- toxicant is meant a substance whose toxicity is known to be of concern, because it can damage organisms when present at anticipated levels.
- biotarget is meant a composition which reacts with at least one toxicant.
- the biotarget may itself have one or more probes or indicator components . When the biotarget reacts with a toxicant, the biotarget indicates an observable change or affects a probe.
- Figure 1 shows several examples of biotargets.
- biotarget is the RBC ( Figures 1A and IB) which normally contains the probe hemoglobin.
- the RBC can be stabilized by well known techniques, such as glutaraldehyde treatment.
- Other examples of biotargets are liposomes, exemplified by
- Figure ID shows a multilayered biotarget with each layer containing a different probe; these containing a multiplicity of compositions that respond to toxicants.
- suitable biotargets include but are not limited to lipids, enzymes, and transport and structural proteins, which react with at least one toxicant .
- biotargets are membranes with proteins (See Figure 2A) , membranes without proteins, vesicles (such as indicator-filled liposomes or particles formed from various cell membranes) , biocoatings (such as mucus, glycoproteins, surfactant coatings, fish dermis, and fish mucus) , biomatrices (such as dermal substrate, collagen, keratin, extracellular and intracellular proteins such as RBC skeletal proteins) with and without biocoatings, cells from relevant structures (such as the lungs, mucous membranes, and gills) , tissue or organ equivalents used alone or with multiple probes (such as an epithelial equivalent which comprises an underlying biomatrix to which epithelial cells are affixed and an optional biocoating) .
- proteins See Figure 2A
- vesicles such as indicator-filled liposomes or particles formed from various cell membranes
- biocoatings such as mucus, glycoproteins, surfactant coatings, fish dermis
- FIG. 2B Another biotarget system ( Figure 2B) utilizes a natural or synthetic biomatrix 46 in combination with a cell layer 47 and a biocoating 48, such as a mucous layer and/or a surfactant coating.
- a probe is associated with the biotarget and changes in reaction to a toxicant. The change can be primary, secondary or even tertiary, in that the change is directly or indirectly caused by a reaction with the toxicant.
- a probe can be a fluorophore, a chromophore a chemiluminescing molecule, a naturally occurring region or group on a protein, or a lipid or an enzyme which can be detected or observed. Probes also can include ions and their transport molecules. Probes can include molecules naturally present in the biotarget. Probes also include enzyme activities, measurements of cell viability, and changes in tissue equivalents or models.
- probes protein components and glycoproteins, structural and enzyme proteins, as well as active, transport components. These probes can be part of the biomembrane, biomatrix, or biocoating systems. Transport proteins and enzymes allow passage of components from one side to the other side of membranes and can serve as probes .
- probes are receptor molecules on membrane or biomatrix surfaces. Actual key biotargets and specific toxicants can change activity structure or function.
- reagent is meant a solution containing solutes which are compatible with the biotarget (s) .
- the reagent is optional.
- the reagent may be formulated with the biotarget or probe.
- the biotarget is an enzyme
- the reagent also preferably contains the probe or substrate upon which the enzyme acts.
- the reagent contains buffers .
- detecting a change is meant registering a change in the test system, such as the visible change in the color of the reagent when a probe is affected or released, as for example, when hemoglobin is released from RBC biotargets.
- Observing a change is exemplified by a visible color change, an audible alarm, a measurable change in electrical charge or current, light, temperature, as well as mechanical changes, such as a change in fluid flow rate or spring action, and a variety of other changes which are discussed below.
- the change can be observed by the human eye (color matching) and/or with a variety of equipment, such as a spectrophotometer, radiation counter, fluorimeter, and nephelometer. The observation can be made a one time (“a static change") or at a series of times (“a kinetic change”) .
- waste stream is meant any fluid discharge which potentially contains toxicants, for example, effluents from chemical plants and municipal sewage plants.
- a waste stream can also be inside a facility, such as fluid from an outlet of an individual chemical processing vat.
- generating a signal is meant giving rise to light or an impulse (such as voltage, current or magnetic strength) by which messages or information can be transmitted. When a signal is generated, it can sound an alarm or cause printing or an electronic read-out.
- system is meant a detection system which acts in concert with biotargets to detect toxicants and quantitate changes.
- the system typically comprises multiple biotargets and probes which provide a multiplicity of changes which in turn are used to profile the test fluid in a sophisticated manner.
- Such a system also can mimic an in vivo system, such as the fish gill test .
- the invention relates to bioassay methods of quantitating and identifying the biotoxicity of fluids which potentially contain toxicants.
- test fluids include water and air. If a toxicant is in a solid form, the toxicant can be added to a fluid before assay by this method.
- the present invention provides methods to test aquatic toxicity and pneumotoxicity.
- RBCs as biotargets.
- hemoglobin a red pigment
- the RBC membrane When the RBC membrane is disrupted or altered, hemoglobin, a red pigment, is released and is easily detected and quantitated visually or spectrophoto ⁇ metrically and the RBC becomes a colorless, shapeless ghost. Released hemoglobin can also react with some toxicants and change color, which also can be detected, particularly spectrophometrically.
- the RBC membrane naturally contains a chemiluminescent molecule which acts as a probe. In the presence of a toxicant, the RBC membrane is altered and chemiluminescence changed.
- biomembranes include vesicles fabricated from portions of intracellular and cell membranes.
- intracellular membranes e.g., mitochondrial membrane
- intracellular membranes can be homogenized and assume the shape of small vesicles.
- Liposomes are stable, water-filled spheres consisting of a lipid bilayer and other optional components. Lipids form liposomes by aligning themselves in parallel: The lipids' hydrophilic ends touching the surrounding water and their hydrophobic ends facing each other (similar to the configuration shown in Figure 2A for biomembranes, where the hydrophilic ends (25) align at the number surface and the hydrophobic portions are tucked inside. Liposomes can envelop diverse molecules, probes or other chemicals.
- Liposomes are constructed from many different types of lipids.
- the lipids in turn can be combined with other molecules, including but not limited to proteins, chemical probes and other chemicals.
- Liposomes can be stabilized by cholesterol, for example, to withstand certain toxicants but dissolve in the presence of other toxicant .
- FIG 2A shows a cutaway of the biomembrane of a biotarget such as an RBC.
- a biotarget such as an RBC.
- the polar groups are exposed to the outer water environment and to any toxicant.
- the toxicant causes at least one change in the membrane components, such as binding characteristics, UV spectrum and other characteristics depending on the toxicant's presence, binding or alteration of the membrane.
- the RBC bilayer also has a fluorescent probe 35 imbedded in the lipid bilayer.
- a protein chromophore 40 is an integral membrane protein which changes color with changes in the bilayer or when protein itself is perturbed.
- a surface protein enzyme 45 will have differing activity, depending on the protein lipid interactions and on the enzymes interaction with toxicants. The activity of enzyme 45 can be used to gauge the presence of toxicants. Also, interaction of the toxicant with the RBC membrane produces a change in its natural chemiluminescence.
- the biotargets are arranged in a wide variety of arrays, including one or a series of layers, in a reagent, on a film.
- the arrangement of the biotargets in one or more layers permits the test fluid to interact in a uniform fashion with the biotargets.
- the biotarget layer can be produced in a variety of ways, for example, by prefabrication or by centrifuging a test mixture as described below.
- the biotargets can be deposited on a film to form a thin layer.
- the biotargets can be solubilized in a solution, which can then be poured onto the film.
- the biotargets themselves receive a biocoating, are encapsulated, dispersed in solids, liquids or semisolids in biocoatings or biomatrices.
- the biotarget solutions and reagents are chosen to be compatible with the particular biotarget (s) .
- the biotargets are RBCs
- the solution or reagent has a physiologic osmolality and pH.
- Solutions compatible with RBCs include but are not limited to normal saline, serum albumin, 5% dextrose, plasma preparations, and other protein-containing solutions.
- Reagents and solutions that are compatible with liposomes vary with the content of the liposome.
- Reagents which are compatible with a number of liposome compositions include but are not limited to water, saline, alcohol in water, etc.
- the solution for depositing biotargets optionally contains a dispersant, such as a minor amount of a starch or gel. After water evaporation, the starch or gel holds the biotargets in place.
- a dispersant such as a minor amount of a starch or gel.
- the biotarget optionally has an underlying biomatrix.
- the underlying biomatrix is "skeletal" protein and intracellular stroma.
- the biomatrix consists of extracellular protein such as collagen and elastin.
- the biotarget includes or reacts with one or more probes, which are released or changed when the toxicant affects the biotarget.
- One probe for the RBC is hemoglobin release.
- Other examples are hemoglobin denaturation, fluorescence of proteins in RBC, chemiluminescence and enzyme activity in RBC membranes .
- probes can be placed in the liposomes. Such probes include but are not limited to colored substances, substances which change color, and fluorophores. Enzymes are useful probes, because upon release from liposomes, they react with a substrate and create visible change such as color.
- probes include fluorescein, cholinesterase, dehydrogenase, tetrazolium salts, methylene blue, lactate dehydrogenase, glucose-6-phosphate, ATP, cytochrome C, lipases, proteases or any mixtures of these probes .
- Another biotarget consists of biocoating.
- the toxicant alters, denatures, coagulates or destroys this coating or layer.
- Coatings can be protein-based, protein and surfactant-based, surfactant-based, extracellular matrix-based, collagen, keratin, structural proteins isolated from dermis, epidermis, generated extracellular matrices from macromolecules.
- One model of a biocoating is fish gill mucus for which a synthetic model is constructed of polymerized protein networks made up of gelatin, globulins, plant globulins, solutions and semisolutions or other glycoproteins.
- the biotargets are contained in a housing 15 which also includes two layers of film 10 to form a biotarget plate 1, although only one layer of film may be necessary if a gel dispersant is used.
- the other surface can alternately be a support .
- the film 10 is a thin, porous, semipermeable membrane. It is thin for easy diffusion of the test fluid; however, if the fluid is sufficiently porous, the film can be thicker.
- the porosity of the film is an important characteristic.
- the film must be sufficiently porous to let the fluid potentially containing toxicants pass through at the desired flow rate. The pore size and number are varied to achieve the desired flow rate for the fluid to which the bioassay plate is exposed.
- the film's pores must be smaller than the biotargets.
- the film is relatively nonreactive with the range of toxic substances to which the film is exposed.
- the film is part of the assay system, turning color or otherwise changing when exposed toxicant (s) .
- a holder 15 may be placed around the perimeter of the film. The requirements of the holder are not stringent .
- the holder must be made of a relatively rigid material which will not interfere with the toxicity test. Many plastics are resistant to the toxic materials to be tested and are acceptable for this purpose. Examples of suitable materials include but are not limited to stainless steel, polyvinyl chloride, ABS plastic.
- the film, biotargets and housing are arranged in the shape of a flat spherical or rectangular bioassay plate, as shown in Figure 3A.
- the bioassay plate 1 is a flattened cube with a film 10 edged by a holder 15.
- the holder 15 is shown to actually surround the edges of the upper and lower films 10.
- the biotargets 20 are contained between the upper and lower films 10.
- Such film sandwiches can be made individually or the film sandwiches can be fabricated in a continuous roll from which individual biotarget sandwiches are cut and compressed into the holder.
- Additional embodiments include various combinations of the bioassay plate of Figure 3.
- Figures 4A-C show that a series of the plates of different shapes can be assembled to have series fluid flow.
- Figures 5A, B and C show different shapes for the bioassay plates, and there are spaces between the plates to permit easy circulation of fluid around both surfaces.
- Figures 6A and B show rectangular bioassay plates 1 assembled into a series (A) and inserted into a partial cartridge 50 (B) .
- Figures 7A and B show a series of spaced bioassay plates (A) inserted into a partial cartridge 50 (B) .
- Figure 8A shows spaced bioassay plates 1 inserted into a carrier 55 with slots 60.
- partial cartridge 50 has upper and lower tracks 65 to slide plates into the cartridge 50 on one side. This arrangement permits the selection of plates which can perform a number of different tests .
- FIGs 9A and B are schematics of cartridges 50 which indicate arrangements of the bioassay plates 1 and fluid flow around the bioassay plates.
- the bioassay plates are generally parallel to the fluid inflow and outflow direction and are spaced to permit maximal flow.
- the bioassay plates are perpendicular to the fluid inflow and outflow and are supported by strips 70 which separate the bioassay plates from the lateral surfaces of the cartridge 50; flow is more turbulent in this configuration.
- FIGS 10A and B show the same parallel and perpendicular arrangements of the bioassay plates but additionally illustrate tracks into which the bioassay plates are inserted.
- the tracks are formed by placing feet to the sides of each bioassay plate.
- Bioassay plates can be assembled into a cartridge, or other experimental design, which are used to perform multiple assays.
- This multifunctional cartridge replaces current in vivo bioassays such as live fish assays, in which the gills are removed and examined or tested for such parameters as biomembrane transport, cell viability, and enzyme activity.
- each plate can be the same or different, in that the plate includes a specific set of biotargets or other types of assay system that are the same or different from those in the other plates. Although only a few plates are shown in Figures 4-10, any number of plates can be used.
- a single biotarget such as RBC may have several, e.g. 20 or more, different probes to provide a toxicity profile of the toxicant or toxicants.
- the combination of plates or assays can be varied for the type of test fluid to be assayed, that is, the expected toxicants.
- heavy metals such as chromate, copper, cadmium, selenium and zinc, are assayed by analyzing protein alterations.
- Organics, such as PCB and pesticides are assayed by their effects on other compositions by testing membrane fluidity and other factors, while other compositions can assay surfactants or detergents.
- compositions, contained in one or more biotargets can be optimized for a particular toxicant that is known to occur in the particular type of test fluid.
- a particular toxicant that is known to occur in the particular type of test fluid.
- very specific antibody tests can be used to identify dioxin
- specific receptors can be used to identify hormones such as insulin and growth hormones.
- the fish gill appears to be the principal target organ for many toxicants.
- a number of classes of toxicants disrupt the barrier properties of the gill epithelium leading to epithelial cell death.
- Fish death usually results from a combination of ionoregulatory, osmoregulatory and respiratory dysfunction.
- the fish epidermis could be a suitable model to determine irritation by toxic compounds, to study the toxicological mechanisms of pollutants, and to assess toxicants.
- Enzyme activity e.g. electron transfer, aldolase, phosphatase, ATPase, etc.
- compositions are conveniently disposed on bioassay plates which can be arranged in a specific or nonspecific order as shown in Figs. 4-10. This combination of tests mimics the behavior of fish gill. Lung tissue can be mimicked with a primary biomembrane equivalent or model, a series of biotargets from subcellular particles, including cellular and living tissue with or without biocoating.
- the series of compositions, also conveniently disposed on bioassay plates has tests for one or more of the following:
- Structural integrity of cell membranes can be measured by RBCs or color-filled liposomes, either of which release a color probe when their membrane structural integrity is damaged.
- Enzyme activity may employ a pulmonary or other enzyme which when activated by a ' toxicant causes a color or other observable change.
- Coating disruption of pulmonary surfaces employs the biotargets coated with pulmonary surfactant or a substitute, wherein disruption of the coating causes the biotargets to release a probe.
- Transport enzymes are inactivated by toxicants to convert measurably less of its substrate.
- Cells from or related to those found in fish gill epithelium or other epithelial structures are used directly.
- Cell viability can be tested with a variety of cells such as an array of cultured cells in a biomatrix as disclosed in U.S. Patent No. 4,963,489.
- Microcalorimetry can be linked to an electrical probe and/or by liquid crystal display. Alternately, key structures can be observed for change associated with temperature changes .
- Additional useful biotargets include fibroblast cells grown from fish or lung or mixed tissue cells grown from lung or gill or mucosa. These are not transplants but are obtained from stable lines of cultures cells.
- bioequivalent of the fish gill, lung, mucosal epithelial or other epithelial equivalents can be used alone or in conjunction with the other biotargets.
- biotargets can be in discs of film, enclosed, encapsulated, coating the film, or on extracellular matrices.
- bioequivalent cultures can be attached to plates or placed in cuvettes, discs and films.
- Figure 11 shows another embodiment of the invention.
- the chamber is filled with a fluid such as air, which the pump 140 moves into the cartridge 50 which contains multiple plates 1 with a diversity of biotargets as described above. After air circulates through the cartridge, it recirculates through a pipe 145 into the air chamber 140.
- the air need not be recirculated but optionally can be recirculated with low level toxicants to predict the toxicants' cumulative irritancy, acute irritancy and other long- and short-term effects.
- Figure 12 is shown the bioassay plate with 2 layers of film 10 holding biotargets 20, mounted in a vial or well. Optionally one layer of film 10 can be used if the biotargets 20 are held in place by a gel.
- Figure 12 also depicts the general appearance of a well in a 24- (or other convenient number) well plate.
- the test fluid is applied to the top film layer and is permitted to diffuse into the biotarget area.
- the toxicant reacts with the biotargets which in turn act on probes which pass through the lower film and into the chamber 147 below the film where the change (s) is detected.
- FIG 13 there are several bioassay plates which can be used to determine the same or different toxicities. If the plates are the same, occurrence of a change in chamber 147 is timed and happens quicker with more toxic substances. If the bioassay plates are different, multiple probes can be detected in chamber 147 by various methods (e.g., spectrophotometric analyses at different wavelengths) .
- the biotargets are mounted on paddles 150.
- One end of a long narrow strip 155, for example, of wood or cardboard is the handle 155, while the other end of the strip is coated with at least one type of biotarget 160.
- the coated end of the paddle can have more than one type of biotarget.
- An example of multiple biotargets is shown in Figure 14B.
- Three types of biotargets 165, 170, 175 are on the top surface of the strip, while the opposing surface has an additional type of biotarget 160.
- the biotargets are coated with a biocoating 180, such as described above. In addition, the biocoating can be covered by the type of film 185 described above.
- This paddle is adapted to field use, for dipping in streams and ponds. It is removed from the fluid and can be immediately read, or the paddle can be rinsed off before the biotargets are observed for a change.
- a surfactant such as 0.1% (w/v) Lubrol PX (polidocanol, available from ICI) may need to be added. Collect the liposomes by centrifugation at 300,000 g for 30 min.
- An alternate method or preparing liposomes is the freeze-thawing technique, which uses detergents. Detergent-free protein is combined with an aqueous dispersion of lipids. The mixture is sonicated or quick- frozen in liquid nitrogen, thawed and sonicated.
- Fibroblast cells from fish gills or other sources are tested for viability and enzyme activity after exposure to toxicants.
- Gill tissue is removed from the fish and sterilized in a solution containing antibiotics. This tissue then is minced under sterile conditions and kept in an appropriate cell culture medium. Next the tissue is centrifuged and supernatant is discarded. The pellet is resuspended in trypsinization medium in a flask with a magnetic stirrer. The tissue is allowed to sediment by gravity. The supernatant containing dissociated cells is drawn off, centrifuged and resuspended in proper medium for further propagation. Derived cells are kept in a C0 2 incubator at appropriate temperature and C0 2 concentration. Alternately, cultured or isolated fibroblasts can be cryopreserved.
- the cells are thawed and mounted on bioassay plates between two layers of film (or within an agar or biomatrix coating) and used to test for toxicants.
- the cells can be grown up on a biomatrix 46 such as agar or an extracellular matrix.
- the cells have a biocoating 48, such as a mucus-like substance.
- any of the following parameters of cell viability and activity can be determined: cell growth, neutral red uptake (NRU) , neutral red release (NRR) , enzyme activity (e.g., ATPase activity, aldolase activity) , gene stress and cell growth numbers.
- a living epithelium equivalent or model is prepared by culturing fish cells 47, such as fibroblasts, mucous cells, or epithelial cells alone or with other stromal cells on a biomatrix 46 scaffolding material, such as fish gill dermis substrate, or other dermal substrate, such as collagen or keratin.
- the epithelial equivalent can be covered by a biocoating 48 or extracellular matrix or coating.
- tissue cultures are mounted in bioassay plates between two films or on a biomatrix. Tests for Surfactant
- Figure 13 shows a schematic for before (A) and after (B) surfactant exposure.
- Figure 13A shows a container
- the container has electrodes 195 on either side which are wired to a meter 200.
- a solution with a low concentration of chloride ions 210 is placed below the bilipid membrane 205 below the bilipid membrane 205 below the bilipid membrane 205 below the bilipid membrane 205 .
- An electrical charge also can be created using differences in osmotic pressure across a matrix or membrane. Perturbation of the biomatrix or biomembrane can cause changes in resistance or be measured as ion flow across the membrane, or increased current flow.
- Another assay measures the electrical potential across a wire that is initially insulated or coated with a substance, such as keratin, which is dissolved off the wire ' by a surfactant. When the keratin insulation dissolves, a change in the electrical potential across the wire is recorded.
- a substance such as keratin
- Another embodiment is a wire embedded in the biomatrix, biomembrane, or biocoating. Upon destruction of the biotarget, the change in electrical potential is detected.
- Another surfactant test utilizes a screen with a biocoating. As test fluid flows through the screen, toxicant dissolves the biocoating. Fluid flow through the screen increases, or the pressure drop across the screen decreases.
- the primary targets are proteins and particularly negatively charged proteins. Heavy metals will affect biotargets, such as proteins or enzyme components, and can cause cell death. Therefore, a change in protein transport, enzyme activity, hemoglobin denaturation, biomatrix and biocoating aggregation indicate the presence of heavy metals.
- Figure 14 shows a biocoating at the top which is a regular matrix of tritiated protein.
- the biocoating Upon alteration by heavy metal-containing fluid, the biocoating has the irregular appearance of the lower half of Figure 14.
- the protein is broken into short strands soluble in water.
- the fluid over the biocoating can be tested for tritiated proteins. Because the biocoating itself is altered and irregular, it will not reflect as much light as the untreated coating: Altered light absorbance also can be observed.
- a cartridge to profile heavy metal contamination is assembled of the following types of biotargets :
- Coating denaturation Lipid fluidity ATP inhibition hemoglobin denaturation cell death Each bioassay plate is read for a change . The combination of presence or absence of changes in the bioassay plates is a profile for the toxicant. If there is mercury in the water, the following are observed: coating denaturation no lipid fluidity change ATP inhibition hemoglobin denaturation cell death
- a membrane with a chlorine pump can be used to detect the effect of the test fluid on the pump.
- the chloride ion concentration can be read by gas chromatography (GC) , high pressure liquid chromatography (HPLC) and spectrophotometer. The change in chloride gradient over time indicates deterioration of the biomembrane' s chloride pump.
- the cartridge contains several plates with the same biotarget Cl- pump membrane.
- the effluent, or test fluid contacts all plates in the cartridge. Individual plates are removed from the cartridge at predetermined times . Chloride ion concentration and gradient are determined. Malfunctioning chlorine transport determined by this method is the foremost cause of fish death and is called bluefish gill syndrome. The fish chloride pump also is affected by low levels of chronic exposure.
- G-6-P Assay for Enzyme Activity Glucose-6-phosphatase (G-6-P) is an enzyme in the
- RBC membrane When the RBC membranes are exposed to toxicants, the toxicant can react with G-6-P and decreased G-6-P activity can be detected. In the presence of nontoxicants, G-6-P activity is not changed.
- the cholinesterase enzyme is incorporated into lyposomes when they are prepared as discussed above. Cholinesterase is specifically inhibited with pesticides which are chlorinated hydrocarbons. Inhibition of this enzyme can be detected as decreased production of the end-product or lack of a change in the concentration of the enzyme's substrate.
- An bioassay plate is constructed to test detergents. The bioassay plate has RBCs between two films which are sufficiently porous to let in detergents but not to let out hemoglobin.
- the detergent-sensitive bioassay plate is exposed to a test fluid suspected of containing detergent . After a sufficient time, the bioassay plate is removed from the test solution and is observed for the presence of released red or denatured brown hemoglobin.
- Bioluminescent bacteria are sensitive to a variety of toxicants. Bioluminescent bacteria are available freeze-dried and are rehydrated when required for use. One such type of luminescent bacteria is Photobacterium phosphoreum B-NRRL 11177 (available from Microbics Corp., Carlsbad, CA) and which is discussed in Bulich et al . , J. Bioluminesc. Chemiluminesc . (1990) 5_:71-77.
- freeze-dried bacteria can be placed between the two films of an bioassay plate of the present invention.
- the bacteria can be accompanied by other chemiluminescent- activated dyes as well as stabilizing agents, including gels.
- the plate is placed in the test fluid for a standard period of time. The time varies with the permeability/flow characteristics of the film.
- the luminescence after a standard time is compared with the luminescence of a control bioassay plate which is not exposed to toxicants .
- This assay utilizes the bioassay plate format described above. The difference here is that between the two layers of film is a complex tissue culture raised from the esophagus of the New Zealand black flounder Rhombosolea retiaria. Shephard, Embryol. Zool . (1982) 5.5:23-34.
- microelectrodes sensitive to sodium, potassium and calcium cations are used to measure the gradients of ion activity within the mucous layer which lies on top of the tissue culture. The electrodes are connected to a recording apparatus, and their potential is measured relative to a grounded electrode.
- Signals from the electrodes can be amplified by a variety of high-impedance instruments, such as a Radiometer electrometer (PHM62) and an Analog Devices 311K.
- high-impedance instruments such as a Radiometer electrometer (PHM62) and an Analog Devices 311K.
- PPM62 Radiometer electrometer
- Analog Devices 311K Analog Devices 311K.
- the very high impedance of the calcium-sensitive electrodes may be overcome by using an I.L. picometric amplifier
- An bioassay plate is prepared from two layers of film, preferably cellophane, between which there are bacteria.
- the bacteria are selected for their susceptibility to a variety of toxicants, including benzene, nitrotoluene, chlorinated benzenes, glycerine, aldehydes, cresols, petroleum products such as kerosene and phenols.
- Suitable bacteria include pseudomonas, bacillus, aerobacter, azotobacter, arthrobacter, micrococcus and nocardia.
- the bacteria are provided in dry powdered form.
- N-fixing, N-cycle bacteria are used to test the aquatic toxicity of effluents in sewage treatment plants, industrial pond sites, and the fermentation of organic components in water.
- the dried cultures are mixed with a dry, powdered gel which contains probe dyes which change color when the bacteria die.
- Suitable gels include but are not limited to agar, starch and gelatin.
- Suitable probe dyes include but are not limited to fluorescein, tetrazolium salts, methylene blue, or any mixtures of these dyes.
- the bioassay plate In use, the bioassay plate is placed in a sample of sewage or other waste water. The plate is removed after a sufficient time for toxicants to diffuse into the bacteria-gel layer and cause a significant change, most desirably about 15 minutes to 3 hours, although the time interval can be varied, depending on the parameters of the film chosen.
- a set of bioassay plates such as depicted in Figures 4 through 10 can be chosen to assay major toxicities of toxicants. By the selected effects, it can be determined which classes of toxicants are present.
- One set of assays for biotargets for profiling includes the following: biomembrane integrity protein conformation membrane fluidity transport activity dehydrogenase activity electron transport function
- biomembranes are lined with at least one probe.
- the layered configuration generally follows
- FIG. 2B This is accomplished by 1) covering a biomatrix 46 with a layer of cells 47 and an overlying biocoating 48 such as mucus, or 2) selecting probe molecules which are hydrophobic and capable of associating with a lipid bilayer. These liposomes contain the probe molecule (s) . Then any exposed probe molecule (s) are stripped off the exterior, so that the test kit only offers liposomes with internal probe molecules. Other methods of coating probe molecules on lipid bilayers are known to those in the art.
- cholinesterase is a suitable probe.
- a pesticide toxicant destroys the bilayer and detaches cholinesterase, cholinesterase is released and free to react with its substrate.
- This test also can be used to identify or help profile cationic heavy metal poisoning because the heavy metals also poison an enzyme such as horseradish peroxidase.
- a solid support such as a stick or long narrow piece of cardboard, is coated with at least one type of biotarget 160, followed by application of a suitable biocoating 180, such as a lipid bilayer.
- a solid support is coated with four biotargets, such as enzymes 165, hapten antigens 170, dye substrates 175 and fluorophores 160.
- the biotarget molecules are covalently bound to the support, which will permit the support surface to be assayed by the appropriate technology.
- a biocoating 180 such as a lipid bilayer.
- the coated support is exposed to a test fluid for a standardized amount of time. The biocoating is destroyed.
- the change in the biotarget molecule can be visually observed by fluorescence, luminescence and/or chemiluminescence.
- the exposed biotarget molecules cause a reaction or response to the enzyme substrate or other suitable molecules .
- the amount of destruction of the biocoating is an indication of toxicant (s) level.
- the profile test utilizes cartridge containing a series of bioassay plates, whose geometric configuration is not important (can be any of Figures 4 through 10) .
- the plates contain the following types of bioassays: intestinal biocoating equivalent biomembrane alteration model mucosal cell equivalent blood partition coefficient model cellular toxicity model
- Each bioassay plate is exposed to a range of concentrations of the test toxicant, new drug, new food additive, etc.
- concentrations range from putatively nontoxic to highly toxic. Positive and negative controls of known safe and unsafe substances are tested in parallel with the unknowns.
- the scores with the various profiles can be used to develop a LD 50 , which is correlated with in vivo toxicity. This also highlights which organ systems might be expected to have the most damage.
- the blood partition coefficient demonstrates the affinity of the toxicant or drug for aqueous and lipid compartments. The first site of transport for poisons is often across the intestinal ucosa and into the blood and then into target organs . If sufficient exchange occurs, the toxicant is removed from blood but is lipophilic and concentrates in the fat.
- Bioassay plates are wired to a central location where changes in electrical current are recorded on strip charts. Excessive changes in current set off audible alarms.
- a central location receives the input and can dam or redirect the toxicant fluid or can levy fines based on the changes recorded at the central location.
- Figure 17 shows a portion of the industrial area of a city's sewer system. Letters A through F represent the various facilities capable of passing toxicants into the system. Circled V s represent valves which can alter sewage flow. Numbered circles 220 through 235 indicate the locations of cartridges of wired bioassay plates which send signals to the city's primary sanitation plant.
- Cartridges 220-230 are located to help identify the source of the pollution. For example, if cartridge 225 is signalling, but not cartridge 220, B is polluting; if cartridge 230 signals, but not cartridge 225, A is polluting.
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Abstract
Un procédé, qui permet d'effectuer un dosage biologique d'un fluide contenant d'éventuelles substances toxiques, consiste à mettre plusieurs compositions en contact avec ce fluide et à détecter au moins un changement, ce qui donne un profil de substance toxique. On décrit aussi une trousse permettant d'appliquer ce procédé afin d'identifier des substances toxiques ou leurs classes grâce à un profil de toxicité basé sur de multiples dosages biologiques.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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AU80950/94A AU8095094A (en) | 1993-10-29 | 1994-10-28 | In vitro fluid toxicity bioassay |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US14476693A | 1993-10-29 | 1993-10-29 | |
US08/144,766 | 1993-10-29 |
Publications (1)
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WO1995011989A1 true WO1995011989A1 (fr) | 1995-05-04 |
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ID=22510038
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1994/012402 WO1995011989A1 (fr) | 1993-10-29 | 1994-10-28 | Dosage biologique in vitro concernant la toxicite d'un fluide |
Country Status (2)
Country | Link |
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AU (1) | AU8095094A (fr) |
WO (1) | WO1995011989A1 (fr) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997012241A1 (fr) * | 1995-09-25 | 1997-04-03 | Gff | Procede et kit de biotest pour la caracterisation d'un support de production vegetale |
EP1066402A1 (fr) * | 1998-02-23 | 2001-01-10 | Phylonix Pharmaceuticals Inc. | Procedes de criblage destines a determiner l'activite d'agents au moyen de poissons teleosteens |
WO2001009041A2 (fr) * | 1999-07-20 | 2001-02-08 | Lockheed Martin Corporation | Systeme de protection d'alimentation en eau de batiments, contre des agents de guerre chimiques et biologiques |
WO2001090402A1 (fr) * | 2000-05-24 | 2001-11-29 | Phplate Stockholm Ab | Nouveau procede et nouveau dispositif |
US6489132B1 (en) * | 1996-10-02 | 2002-12-03 | Safety Associates, Inc. | Methods and apparatus for determining specific analytes in foods and other complex matrices |
WO2003008935A2 (fr) * | 2001-07-20 | 2003-01-30 | Bols Niels C | Procede et dispositif d'echantillonnage et de toxicovigilance |
US7482507B2 (en) | 1998-02-23 | 2009-01-27 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7767880B2 (en) | 2006-09-01 | 2010-08-03 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
FR3078075A1 (fr) * | 2018-02-21 | 2019-08-23 | Centre National De La Recherche Scientifique (Cnrs) | Methode d'estimation de la toxicite globale d'un echantillon aqueux, kit pour sa mise en œuvre et utilisation pour divers types d'effluents. |
CN110346342A (zh) * | 2019-08-28 | 2019-10-18 | 苏银华 | 一种农药残留检测方法 |
CN113820376A (zh) * | 2021-09-14 | 2021-12-21 | 南开大学 | 一种基于机器学习模型的微生物电化学传感器的综合毒物监测方法 |
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US4835102A (en) * | 1987-03-31 | 1989-05-30 | Eugene Bell | Tissue equivalent test systems |
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- 1994-10-28 AU AU80950/94A patent/AU8095094A/en not_active Abandoned
Patent Citations (1)
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US4835102A (en) * | 1987-03-31 | 1989-05-30 | Eugene Bell | Tissue equivalent test systems |
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ANNALS OF NEW YORK ACADEMY OF SCIENCES, issued 1983, BJORN EKWALL, "Screening of Toxic Compounds in Mammalian Cell Cultures", pages 64-77. * |
Cited By (26)
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CH689464A5 (de) * | 1995-09-25 | 1999-04-30 | Jacques Fuchs | Verfahren und Biotest-Kit zur Charakterisierung eines Pflanzenproduktionstraegers. |
WO1997012241A1 (fr) * | 1995-09-25 | 1997-04-03 | Gff | Procede et kit de biotest pour la caracterisation d'un support de production vegetale |
US6489132B1 (en) * | 1996-10-02 | 2002-12-03 | Safety Associates, Inc. | Methods and apparatus for determining specific analytes in foods and other complex matrices |
US7951989B2 (en) | 1998-02-23 | 2011-05-31 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US7482507B2 (en) | 1998-02-23 | 2009-01-27 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
US8993834B2 (en) | 1998-02-23 | 2015-03-31 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
EP1066402A4 (fr) * | 1998-02-23 | 2001-06-13 | Phylonix Pharmaceuticals Inc | Procedes de criblage destines a determiner l'activite d'agents au moyen de poissons teleosteens |
EP1066402A1 (fr) * | 1998-02-23 | 2001-01-10 | Phylonix Pharmaceuticals Inc. | Procedes de criblage destines a determiner l'activite d'agents au moyen de poissons teleosteens |
EP1548123A1 (fr) * | 1998-02-23 | 2005-06-29 | Phylonix Pharmaceuticals Inc. | Procédés de criblage destinés a determiner l'activité d'agents au moyen de poissions téléosteens |
US7687682B2 (en) | 1998-02-23 | 2010-03-30 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
EP1878799A2 (fr) * | 1998-02-23 | 2008-01-16 | Phylonix Pharmaceuticals Inc. | Procédés de criblage d'agents pour l'activité utilisant des téléosts |
EP1878799A3 (fr) * | 1998-02-23 | 2008-01-23 | Phylonix Pharmaceuticals Inc. | Procédés de criblage d'agents pour l'activité utilisant des téléosts |
US7435870B2 (en) | 1998-02-23 | 2008-10-14 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
WO2001009041A3 (fr) * | 1999-07-20 | 2001-10-04 | Lockheed Corp | Systeme de protection d'alimentation en eau de batiments, contre des agents de guerre chimiques et biologiques |
WO2001009041A2 (fr) * | 1999-07-20 | 2001-02-08 | Lockheed Martin Corporation | Systeme de protection d'alimentation en eau de batiments, contre des agents de guerre chimiques et biologiques |
US8551921B2 (en) | 2000-05-24 | 2013-10-08 | Phplate Stockholm Ab | Method for determining the toxicity of an environmental or chemical sample |
WO2001090402A1 (fr) * | 2000-05-24 | 2001-11-29 | Phplate Stockholm Ab | Nouveau procede et nouveau dispositif |
US7186565B2 (en) | 2001-07-20 | 2007-03-06 | Kristin Schirmer | Sampling and toxicity monitoring device and method |
WO2003008935A3 (fr) * | 2001-07-20 | 2003-08-28 | Niels C Bols | Procede et dispositif d'echantillonnage et de toxicovigilance |
WO2003008935A2 (fr) * | 2001-07-20 | 2003-01-30 | Bols Niels C | Procede et dispositif d'echantillonnage et de toxicovigilance |
US7767880B2 (en) | 2006-09-01 | 2010-08-03 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
FR3078075A1 (fr) * | 2018-02-21 | 2019-08-23 | Centre National De La Recherche Scientifique (Cnrs) | Methode d'estimation de la toxicite globale d'un echantillon aqueux, kit pour sa mise en œuvre et utilisation pour divers types d'effluents. |
WO2019162619A1 (fr) * | 2018-02-21 | 2019-08-29 | Centre National De La Recherche Scientifique | Methode d'estimation de la toxicite globale d'un echantillon aqueux, kit pour sa mise en œuvre et utilisation pour divers types d'effluents |
CN110346342A (zh) * | 2019-08-28 | 2019-10-18 | 苏银华 | 一种农药残留检测方法 |
CN110346342B (zh) * | 2019-08-28 | 2021-12-21 | 苏银华 | 一种农药残留检测方法 |
CN113820376A (zh) * | 2021-09-14 | 2021-12-21 | 南开大学 | 一种基于机器学习模型的微生物电化学传感器的综合毒物监测方法 |
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