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US20020039725A1 - Fish hatching method and apparatus - Google Patents

Fish hatching method and apparatus Download PDF

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Publication number
US20020039725A1
US20020039725A1 US09/930,499 US93049901A US2002039725A1 US 20020039725 A1 US20020039725 A1 US 20020039725A1 US 93049901 A US93049901 A US 93049901A US 2002039725 A1 US2002039725 A1 US 2002039725A1
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species
hatching
diapause
test
embryos
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Tommy Shedd
Mark Widder
Eugene Hull
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United States Department of the Army
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Priority to US09/930,499 priority Critical patent/US20020039725A1/en
Publication of US20020039725A1 publication Critical patent/US20020039725A1/en
Priority to US10/454,821 priority patent/US7094417B2/en
Assigned to UNITED STATES ARMY reassignment UNITED STATES ARMY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WIDDER, MARK W., SHEDD, TOMMY R.
Priority to US11/340,757 priority patent/US8574603B2/en
Abandoned legal-status Critical Current

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    • 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
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • G01N2520/00—Use of whole organisms as detectors of pollution

Definitions

  • This invention relates to the field of animal husbandry.
  • a rapid hatching fish embryo kit and method for using same is disclosed.
  • the invention can be used in any application where it is desirable to store and/or transport fish embryos that can be removed from the storage media and hatched, preferably within about 48 hours.
  • the present invention can be used to perform rapid toxicity assessments in the field where it would otherwise be difficult to culture the fish necessary to perform such tests.
  • the present invention can be used as a convenient means for distributing fish to investigators, aquaculturists, hobbyists, and the like.
  • these tests systems commonly measure bacterial bioluminescence or respiration (e.g., Microtox, Azur Environmental, Carlsbad, Calif., USA; Polytox, Bethlehem, Pa., USA) and mortality or growth of organisms after emergence from resting life stages (e.g., rotifer cysts, brine shrimp cysts, or lettuce seeds).
  • respiration e.g., Microtox, Azur Environmental, Carlsbad, Calif., USA; Polytox, Bethlehem, Pa., USA
  • mortality or growth of organisms after emergence from resting life stages e.g., rotifer cysts, brine shrimp cysts, or lettuce seeds.
  • Nothobranchius guentheri is an annual killifish indigenous to the coastal lowlands of Kenya and Kenya.
  • the embryonic development of N. guentheri has been extensively studied and is known to proceed through three successive stages, designated as diapause 1 , 2 , and 3 .
  • diapause 1 the embryo is typically characterized as an undifferentiated mass of cells.
  • diapause 2 the embryo is typically characterized as being partially developed and having undifferentiated cells.
  • diapause 3 the embryo is typically characterized as being fully developed and ready to hatch in the presence of water or at the onset of rainfall.
  • a test method has been developed in part to exploit these unique features, using newly hatched killifish ( Nothobranchius guentheri ) for rapid acute toxicity screening.
  • Embryo culturing methods have been established and a storage media has been developed to allow convenient recovery of embryos for testing from storage media. After recovery, the embryos may be hatched from storage for use in a toxicity test.
  • the rapid fish acute toxicity test uses the killifish Nothobranchius guentheri. Recent studies have demonstrated that killifish can be mass cultured to produce large numbers of embryos in a suspended state (diapause). The embryos in diapause can be stored for long periods of time in semi-dry laboratory conditions for future toxicity testing. By changing the killifish embryo holding conditions, the embryos will hatch and the fry can be grown and used for testing. A fish toxicity test system that requires no culture at the time of testing would be especially useful and cost effective. The rapid killifish test could be conducted at any time without concern for culture system requirements to provide fish for testing, nor would conducting toxicity testing be subject to the vagaries of seasonal growth patterns and the delayed time associated with producing suitable test subjects.
  • FIG. 1 is a flow chart illustrating a method and kit of an embodiment of the invention.
  • the present invention is a rapid test for analyzing toxicity comprising exposing a diapause species to a hatching medium, allowing the diapause species to form hatchlings, and exposing the hatchlings to toxicity test materials.
  • the diapause species is a fish species.
  • the fish is an annual fish species.
  • the diapause species is a killifish, even more preferably, from the species Nothobranchius guentheri.
  • the species is in diapause 3 .
  • exposing a diapause species to a hatching medium includes a hatching medium comprising at least one of the following: water, ground water; any form of water that is non-toxic to the species being tested; and any form of water containing one or more nutrients and/or minerals.
  • Typical minerals include, but are not limited to, inorganic minerals or salts, such as calcium, potassium, and/or magnesium containing compounds. Typically this is sufficient for a rapid or short term toxicity test.
  • longer term tests e.g., those that involve growth or a longer life span of the test species, may require other ingredients suitable for maintenance and survival of the species.
  • Some embodiments of the invention further involve obtaining a species in diapause 3 .
  • the present invention is also a method for testing the toxicity of a test material comprising exposing a test sample to hatchlings from a species that includes a diapause state, and determining the effect of the test sample on the hatchlings. Some embodiments of the invention further involve obtaining a species in diapause 3 .
  • the present invention is also a method for distributing a species comprising obtaining a species in its diapause state, storing the species in a storage medium, shipping the storage medium containing the species to a pre-determined location, separating the species from the storage medium, and exposing the species to a hatching medium.
  • Some embodiments of the invention further involve obtaining a species in diapause 3 .
  • Some embodiments of the present invention also include a storage medium for a species in its diapause state.
  • a storage medium according to the present invention comprises a composition suitable for maintaining a diapause species in its diapause state.
  • a process in accordance with the invention also may include the hatching medium capable of inducing growth and proliferation of the diapause species.
  • hatching medium is used to describe a medium, such as a nutrient medium, that favors rapid growth of the species.
  • the present invention may also include a hatching apparatus comprising a container having a closed end and an open end, said closed end being shaped to position embryos placed therein in close proximity.
  • a hatching apparatus comprising a container having a closed end and an open end, said closed end being shaped to position embryos placed therein in close proximity.
  • the shape of the closed end is conical.
  • the open end further comprises one or more arcuate notches or the like.
  • the present invention also includes a kit for conducting a toxicity test, comprising a composition comprising a species in its diapause state and a storage medium, one or more containers for hatching the species, and one or more containers for exposing the hatchlings to a test material.
  • the kit may also include one or more of the following: a container for storing the composition; a filter or the like for separating the species from the storage medium; a hatching medium; one or more growth factors or promoters suitable for adding to the hatching medium; one or more containers for culturing and/or growing hatchlings.
  • Some embodiments of the invention further involve obtaining a species in diapause 3 .
  • the present invention also includes a kit for conducting a toxicity test in accordance with the present invention, in combination with the devices and ingredients of other toxicity tests.
  • diapause species refers to any living organism or cell having a diapause state.
  • Exemplary diapause species include, but are not limited to, certain fish, chickens, almost all insects, and frogs.
  • the diapause species may be an animal, plant, or microorganism species.
  • Diapause state refers to a condition of suspended development or any pause occurring in the process of embryonic development.
  • a diapause state may be induced or may be in response to an environmental condition.
  • An example of inducing a diapause state may include mediating the diapause state using one or more diapause factors.
  • One or more diapause factors may be included in or added to the solution containing the diapause species, and the factors may up-regulate or down-regulate the biological processes associated with entering diapause, maintaining diapause, and/or transitioning out of diapause.
  • An example of a response to an environmental condition may include a response to a drought condition, in which some fish species produce drought-resistant eggs that are capable of entering diapause, and remain in diapause until a sufficient amount of water or rain contacts the eggs.
  • the diapause species is a fish species.
  • the fish is an annual fish species.
  • the diapause species is a killifish, even more preferably, from the species Nothobranchius guentheri.
  • storage medium refers to any medium or composition suitable for storing and maintaining a species in its diapause state.
  • the storage medium maintains or controls the moisture content in a predetermined range suitable for the diapause species, and keeps individual embryos separated.
  • a storage medium may be selected according to one or more of the following qualities: even insulative properties, maintaining a relatively constant humidity, ease of separation from the diapause species, non-toxic to the diapause species, and maintaining separation between individual embryos.
  • Typical compositions may include at least one of the following: peat moss, refined peat moss, filter paper, processed paper and/or paper products.
  • Water may also be used as a storage medium, but a container or structures to maintain separation between individual embryos should be used to maintain the embryos in diapause.
  • Filter paper is preferred for short term storage; refined peat moss is preferred for long term storage, in part due to the ease of maintaining environmental conditions conducive to diapause embryo survivability.
  • the storage medium has a water content above about 50% by weight, preferably above about 75% by weight, up to about 100% by weight.
  • the most preferred storage medium is refined peat moss, due in part to the ease of maintaining environmental conditions conducive to diapause embryo survivability, and the ease with which the diapause embryos may be separated from the medium when ready for use.
  • peat moss may be refined by processing or filtering the peat moss to achieve a uniform particle size, typically less than about 0.6 mm (or a size that is smaller than the typical embryo size).
  • hatching medium or hatching refers to any medium or composition suitable for inducing a diapause species to transition out of its diapause state. Typically, the hatching medium will stimulate embryo growth.
  • Typical hatching media in accordance with the invention may include water alone, or water including a carbon source, a source of inorganic ammonia (or ammonium ion), a source of phosphate, one or more hormones or animal/plant growth regulators, and optionally, other nutrient sources.
  • exemplary hatching medium includes but is not limited to water, deionized water, any form of water containing one or more growth factors or growth inducers.
  • the storage medium and/or the hatching medium may include one or more salts to sufficient to reduce or eliminate fungal growth.
  • the hatching medium is deionized water.
  • the content and other attributes of the water may be altered for a specific species or to achieve a specific result. Any additive to the medium that promotes and/or enhances survivability of the species may be used in the hatching medium. These alternative compositions are included in the present invention.
  • the pH of the water is from about 6 to about 8; alkalinity of the water is typically from about 15 to about 35 mg/L (measured as CaCO 3 ), and the hardness of the water is typically from about 30 to about 50 mg/L (measured as CaCO 3 ).
  • water having a pH from about 7.75 to about 7.82, alkalinity from about 20 to about 28 mg/L (measured as CaCO 3 ), and hardness from about 40 to about 44 mg/L (measured as CaCO 3 ) has been found suitable for growth of Nothobranchius guentheri.
  • the hatching stage may occur under other various conditions. Conditions such as pH, temperature, light, and time may vary for different species in order to optimize cell growth. Further, it may be desirable to replace used hatching media with fresh hatching media since enzymes produced as a function of the hatching process may be toxic. For example, it may be desirable to transfer hatchlings from a hatching tube to a hatching dish, or similar larger container, in order to minimize or eliminate the effect of these enzymes.
  • hatching may include one or more steps or structures that promote or facilitate embryo contact or proximate positioning sufficient to induce or promote hatching.
  • toxicity test refers to any test in which a species is contacted with a composition and evaluated for any deleterious effects from an ingredient in the composition.
  • the hatching medium and the test medium are the same or include the same ingredients, except that the test medium further includes the one or more potentially toxic elements being tested.
  • Diagnostic microbiology and toxicology typically base diagnoses on a macroscopic and/or microscopic examination of a living organism. The accuracy of the diagnosis and the preparation of optimally interpretable specimens typically depends upon adequate sample preparation, i.e., the rapidity with which a potentially toxic test sample can be placed in contact with a test organism.
  • a rapid toxicity test is defined in this application as a test that is 96 hours or less in duration, uses standard laboratory equipment, and has no continuous culture requirements to provide test organisms. Typical rapid toxicity tests are for about 24 hours or about 48 hours in duration.
  • An exemplary time frame for Nothobranchius guentheri includes about 1 to 2 hours for initial hatching, about 24 hours for optimum hatching, and about 24 hours for exposure to a test composition containing one or more toxins.
  • the killifish test can be used in combination with other rapid tests to give a multi-tropic-level assessment at sensitivity levels near the standard EPA tests. Savings in culture and testing resources can be applied for a more complete contaminant or on-site screening evaluation to assess potential toxic impact on the environment.
  • Other exemplary tests include, but are not limited to those described by the U.S. Environmental Protection Agency and acceptable toxicity tests. For example, the EPA has published “Methods of Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms” (fourth edition), EPA/600/4-90/027 (September 1991), incorporated herein by reference.
  • the methods and kits of the present invention provide beneficial superiority over existing toxicity methods because: 1) embryos in diapause can be stored for an extended period at normal room temperature (about 21° to about 25° C.) before use; 2) hatching occurs rapidly, typically within about one to two hours after the embryos are placed in water; 3) the storage media maintains embryos in a desired diapause state and simplifies subsequent embryo separation from the storage media; 4) the size and shape of the hatching tubes encourage hatching to occur and reduce handling stress that could harm embryos and hatchlings; and 5) the culturing wells further reduce handling stress.
  • FIG. 1 illustrative concepts of the present invention are shown.
  • the invention as illustrated includes embryos 1 , storage media 2 , container 3 , hatching tubes 4 , culture plate 5 , and culture water 6 .
  • container 3 holds embryos 1 and storage media 2 , typically at normal room temperature (e.g., about 21° to about 25° C.).
  • the diapause embryos can be maintained in this condition for up to about three years or more, any period within 0 to 3 years, and typically nine months or more before use, preferably between about 6 months and about 9 months.
  • storing the diapause embryos in a vacuum pack or on a shaker may extend the storage period.
  • One skilled in the art will also recognize that the embryos for some species may be stored for longer than nine months, but the chances for embryo degradation appear to increase over time.
  • embryos 1 may be removed from container 3 , separated from storage media 2 , and placed into hatching tubes 4 with about 1 mL of the culture water 6 . It has been found that the choice of the size and shape of the hatching tube, the amount of culture water, and the number of embryos in each hatching tube should be coordinated to promote contact among the embryos. This is based on the finding that separated embryos rarely hatch, but that embryos in close proximity to each other hatch, sometimes quickly.
  • the hatching tubes may then be placed into holding or growth cells 7 .
  • the hatching tube is placed in the holding cell 7 so that hatchlings can swim out of the hatching tube into the holding cell, but embryos are retained in the hatching tube.
  • An exemplary configuration is shown in FIG. 1.
  • hatching tube 4 is set at an angle in holding cell 7 .
  • the ability of the hatchling to remove itself from the hatching tube may be enhanced by providing a hatchling tube with one or more arcuate cut-outs 8 .
  • embryos 1 are obtained from an annual fish species, preferably a killifish.
  • the killifish is Nothobranchius guentheri, which is native to the coastal lowlands of Africa and Kenya.
  • Annual fish are unique in that they are common only in habitats which are subject to complete seasonal drying and erratic climatic conditions.
  • Diapause embryos may be found in nature, or may be purchased from Orbis Scientific or Triops, Inc., both companies having a place of business in Pensacola, Fla.
  • the embryos may be stored in a storage medium as noted above.
  • One exemplary storage medium is refined peat moss, peat moss that ground to a particulate rating that will pass through a 0.6 mm sieve. As further illustrated below, grinding the peat sized to a pre-determined particulate rating aids in the separation of embryos from storage media when the user is ready to begin culturing.
  • the storage media is semi-dry.
  • semi-dry refers to the moisture content of the storage medium.
  • the storage medium should sufficiently moist to preserve the embryos. Without intending to be limited to a specific moisture content, the inventors have found that a moisture content between about 50% and about 80% by weight, preferably about 75% percent water by weight, is suitable for preserving N. guentheri embryos.
  • Preferred storage media are those that promote even insulative properties, maintain a constant humidity, allow ease of embryo removal, and is non-toxic to the embryos.
  • the storage media is preferably sterilized before use, e.g., by boiling or autoclaving.
  • the peat moss or other media may be liquified and blended to reduce particulate size.
  • the media may be strained or filtered though sieve 9 or the like, e.g., a fine polypropylene mesh (typically, about 0.6 mm), to remove particles that exceed minimum embryo size.
  • the storage media particulate size should be smaller than the embryos so that the embryos may be separated from the media by straining though the same mesh.
  • excess water may be removed by filtering the media though a brine shrimp net. The net may be lightly squeezed so that the media is not saturated with water but is still moist.
  • compositions may be used as storage media, e.g., filter paper.
  • Choice of storage media may be selected in part according to desired survival rates, as is well known to those skilled in the art.
  • Container 3 is preferably a sealable plastic bag, petri dish, or similar container for holding embryos 1 and storage media 2 during storage and/or transport.
  • embryos 1 may be protected from outside sources of contamination, and the moisture content of storage media 2 may be maintained or controlled.
  • Hatching tube 4 is generally conical in shape with beveled edges 10 - 11 and an approximate volume of 1 cc.
  • the size and shape of hatching tube 4 causes the embryos to cluster near the bottom of the tube such that each embryo is generally in contact with two or more other embryos.
  • contact with other embryos appears to provide beneficial results, including but not limited to stimulating embryo growth.
  • Hatching generally occurs within 1 to 2 hours of embryo placement in the hatching tubes.
  • beveled edges 10 - 11 provide a path for hatchlings to swim free of the hatching tube and into the larger volume of the culture plate. This eliminates handling stress that may result from the use of conventional transfer devices, e.g., a pipette or similar device.
  • Culture plate 5 is a multi-cell test culture plate.
  • Culture water 6 is a soft water suitable for hatching fish.
  • a deionized ASTM Type 1 water may be used.
  • the deionized water includes one or more inorganic salts, or one or more other ingredients used to reduce algae and/or fungal growth, including but not limited to NaHCO 3 , CaCl(2H 2 O), MgCl 2 (6H 2 O), KCl, and NaCl.
  • the culture water and the test medium are the same, preferably both formulated according to conventional ASTM standards.
  • Acceptable water quality parameters for the culture water 6 are: pH (7.75 -7.82), alkalinity (20-28 mg/L as CaCO 3 ), hardness (40-44 mg/L as CaCO 3 ).
  • a contaminant in a fluid such as air or water, e.g., contaminants in drinking water, or bacteria in food and beverage processing plants.
  • a certain contaminant such as estrogenic compounds, pesticides (DDT, heptachlor, and atrazine), aromatic hydrocarbons, and polychlorinated biphenyls.
  • DDT pesticides
  • heptachlor heptachlor
  • atrazine aromatic hydrocarbons
  • polychlorinated biphenyls polychlorinated biphenyls.
  • breakdown products such as bisphenol-A, an ingredient in plastics.
  • the typical acute toxicity test will test for contaminants and the like in environmental effluent, will involve a dilution series of the effluent, and will test for toxicity as the endpoint of the analysis.
  • Dilution water utilized was a deionized ASTM Type 1 water with 48 mg/L NaHCO 3 , 26 mg/L CaCl(2H 2 O), 51 mg/L MgCl 2 (6H 2 O), 2 mg/L KCl, and 2 mg/L NaCl added.
  • Acceptable water quality parameters for the dilution water were as follows: pH (7.75 -7.82), alkalinity (20-28 mg/L as CaCO 3 ), hardness (40-44 mg/L as CaCO 3 ).
  • Enough embryos were shipped via overnight airmail to perform tests (approximately 600 embryos per test chemical). Embryos received for testing were used immediately or placed in long-term storage at 21° C.-25° C. The storage area should be temperature controlled to avoid premature emergence of fry.
  • the storage media consists of filter paper sealed in a petri dish (0 to 60 days) and/or a refined peat moss (particulate size ⁇ 0.6 mm for greater than 60 days storage). Embryos were stored in these semi-humid conditions until needed for testing. The embryos were then extracted from the peat by straining the embryos through a 0.6 mm mesh.
  • the embryos were rinsed from their storage media with dilution water into a petri dish. Temperature was maintained at 25° C. Dead, discolored, and prematurely hatched embryos were not used. Embryos were randomized using a disposable pipette into 24 separate hatching tubes containing 1 mL of dilution water.
  • Storage media containing embryos is saturated with culture water.
  • the culture water should be in the range 20-25° C.
  • Embryos were extracted from the storage media by pouring the liquified storage media through the 0.6 mm mesh. The embryos collected on the mesh were rinsed into a holding dish. Embryos were then transferred from the holding dish into the hatching tubes using a pipette. To achieve a pre-determined number of hatchlings in each cell well, 50% mortality was assumed, so two times the number of embryos were placed in each cell.
  • Hatching tubes containing fry and embryos were transferred into their respective test chambers when hatch was initiated. All hatching tubes were removed from the test wells within two hours of hatch initiation. Unhatched embryos and egg cases were removed from the test well. Fry were examined under the dissecting microscope. Malformed fry were noted and discarded. Remaining fry were incubated at 25° C. without light for a 24 hour period.
  • fry were examined under a dissecting microscope and all dead or malformed fry were removed. Fry were thinned to 10 fry per test well with a plastic disposable pipette.
  • Diapause 3 embryos stored in saline-moistened filter paper were shipped overnight from Triops (Pensacola, Fla., USA) and were used within 24 h of arrival for testing unless noted otherwise. A minimum of 600 embryos per test chemical was used. For long-term storage, embryos were placed in sterile (autoclaved), moist (75% water by weight) peat moss ground to a particular size of ⁇ 0.6 mm. Embryos stored in this medium in the dark at 21 to 25° C. have remained viable for at least 9 months.
  • Culture water was deionized American Society for Testing and Materials type 1 water containing 48 mg/L NaHCO 3 , 26 mg/L CaCl-2H 2 O, 51 mg/L MgCl 2 -6H 2 O, 2 mg/L KCl, and 2 mg/L NaCl; the culture was aerated 24 hours before use. Acceptable parameters were pH 7.75 to 7.82; alkalinity, 20 to 28 mg/L (as CaCO 3 ); and hardness, 40 to 44 mg/L (as CaCO 3 ).
  • Hatching tubes were 1.5-mL disposable polypropylene Eppendorf conical microcentrifuge tubes modified by a bevel cut at the top ( see FIG. 1).
  • the embryos received in filter paper storage, were washed into the hatching tubes with 1 mL of culture water.
  • Embryos in long-term storage were separated by sieving the peat moss through a 0.6-mm polypropylene mesh screen and were similarly washed from the screen into the hatching tubes.
  • the tubes, each containing about 20 embryos, were placed at a 45° angle in test wells containing 10 mL of culture water.
  • the design of the hatching tube allowed the embryos to cluster at the bottom and enabled the hatchlings (fry) to swim freely into the test chambers without the stress of transfer (see FIG. 1). Hatching began within 1 hour, and 2 hours later the hatching tubes and any unhatched embryos and egg cases were removed from the test wells. Hatch rates ranged from 85 to 95% with a mean of 89%. Twenty-four hours after hatch, fry were examined with a dissecting microscope, and any damaged or malformed fry were removed. The fry were thinned to 10 per test well for subsequent toxicity test.
  • Toxicity tests were performed in triplicate at five concentrations (0.5 dilution factor) with control, in the same test wells in which fry were hatched (FIG. 1), in a Labline Biotronette incubator (Melrose Pak, Ill., USA) at 25° C.
  • FOG. 1 Labline Biotronette incubator
  • Nine milliliters of culture water was removed from each well and replaced with 9 mL of the test chemical solution in culture water.
  • the wells were sealed with parafilm covered.
  • Reference tests with zinc as the toxicant were run alongside every test chemical to measure the batch variability of the killifish fry throughout the study. Tests were terminated after 24 hours, and the number of healthy, stressed, and dead fry in each test well were recorded. Healthy fry moved rapidly through the water and had constant, rapid pectoral fin movement.
  • the sensitivity of the killifish test was in a range comparable to that of the other test organisms.
  • the killifish test equaled or exceeded the standard methods in sensitivity, and for 1-octanol, phenol, PCP, 2,4-D, TNT, and SDS, the killifish sensitivities were within a range similar to those of the U.S. EPA test organisms.
  • D. magna is known to be exceedingly sensitive to malathion
  • the killifish endpoints for the malathion were comparable to those observed for P. promelas and M.bahia.
  • the sensitivity of the killifish was somewhat lower than that of the standard test systems but not greatly different from that observed for P. promelas.
  • N. guentheri hatchlings as organisms for acute toxicity assessments has shown them to be comparable in sensitivity and reproducibility to a number of commonly used standard aquatic species in response to 11 representative toxicants, including heavy metals, unionized ammonia, biocides, and other trace organics.
  • a major advantage of this species is that there is no need to maintain a continuous culture to have organisms immediately available for testing. Moreover, diapause 3 embryos remain viable for up to 9 months in moist peat moss and can readily be transported for on-site assessments of pollution.
  • Other advantages include rapidity and simplicity of the assay; EC50 and LC50 endpoints can be determined after 24 h, and only standard laboratory equipment is required.
  • Killifish test was similar in sensitivity to standard fathead minnow test. Killifish EC50's were more sensitive indicators of toxicity than LC50's for all compounds tested. Embryo batch variability in response to zinc exposure was low. EC50's were less variable than LC50's. Long-term storage of embryos did not affect LC50 sensitivity. Overall control mortality was low, ⁇ 10%.

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  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Catching Or Destruction (AREA)
US09/930,499 2000-08-17 2001-08-16 Fish hatching method and apparatus Abandoned US20020039725A1 (en)

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US09/930,499 US20020039725A1 (en) 2000-08-17 2001-08-16 Fish hatching method and apparatus
US10/454,821 US7094417B2 (en) 2000-08-17 2003-06-05 Fish hatching method and apparatus
US11/340,757 US8574603B2 (en) 2000-08-17 2006-01-27 Hatching kit for toxicity test

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US22578800P 2000-08-17 2000-08-17
US09/930,499 US20020039725A1 (en) 2000-08-17 2001-08-16 Fish hatching method and apparatus

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US11/340,757 Expired - Fee Related US8574603B2 (en) 2000-08-17 2006-01-27 Hatching kit for toxicity test

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US11/340,757 Expired - Fee Related US8574603B2 (en) 2000-08-17 2006-01-27 Hatching kit for toxicity test

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US (3) US20020039725A1 (fr)
EP (1) EP1311156A4 (fr)
AU (1) AU2001290535A1 (fr)
CA (1) CA2419555A1 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116267721A (zh) * 2023-04-13 2023-06-23 厦门生命互联科技有限公司 一种抗黑色素生成功效评价方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001290535A1 (en) 2000-08-17 2002-02-25 U.S. Army Medical Research And Materiel Command Fish hatching method and apparatus
FR2883078B1 (fr) * 2005-03-10 2008-02-22 Essilor Int Imageur optique destine a la realisation d'un afficheur optique
ITRM20050169A1 (it) * 2005-04-07 2006-10-08 Lay Line Genomics Spa Uso di nothobranchius furzeri come sistema modello per la caratterizzazione di geni e farmaci che controllano l'invecchiamento.
US20100037829A1 (en) * 2008-08-15 2010-02-18 Binkley Dennis E Easy hatch aquarium
EP2525653A4 (fr) * 2010-01-20 2016-11-02 Childrens Medical Center Procédé et système pour production en masse d'embryons de poissons
DE102014018936B3 (de) * 2014-12-22 2015-12-31 Speetect GmbH Nachweis von giftigen Substanzen und Strahlung mittels eines "Drosophilatoximeters"
CN110326559B (zh) * 2019-08-08 2022-03-25 潘洪强 一种团头鲂鱼苗的繁育方法

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US5593678A (en) * 1994-10-11 1997-01-14 University Of Georgia Research Foundation Protection of teleost fish
US5932436A (en) * 1996-12-06 1999-08-03 Wisconsin Alumni Research Foundation Daphnia reproductive bioassay for testing toxicity of aqueous samples and presence of an endocrine disrupter
US6150126A (en) * 1996-12-06 2000-11-21 Wisconsin Alumni Research Foundation Daphnia reproductive bioassay for testing toxicity of aqueous samples and presence of an endocrine disrupter
AU2001290535A1 (en) 2000-08-17 2002-02-25 U.S. Army Medical Research And Materiel Command Fish hatching method and apparatus

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116267721A (zh) * 2023-04-13 2023-06-23 厦门生命互联科技有限公司 一种抗黑色素生成功效评价方法

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US8574603B2 (en) 2013-11-05
WO2002013598A3 (fr) 2002-03-28
CA2419555A1 (fr) 2002-02-21
EP1311156A4 (fr) 2006-12-06
US20060140862A1 (en) 2006-06-29
WO2002013598A2 (fr) 2002-02-21
US7094417B2 (en) 2006-08-22
AU2001290535A1 (en) 2002-02-25
US20040013608A1 (en) 2004-01-22
EP1311156A2 (fr) 2003-05-21

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