AU2014201934B2

AU2014201934B2 - Composition and method for amphibian euthanasia

Info

Publication number: AU2014201934B2
Application number: AU2014201934A
Authority: AU
Inventors: Donald John Bell; Nicholas Robert Paton; Randal Phillips
Original assignee: AQUI S NEW ZEALAND Ltd
Current assignee: AQUI-S NEW ZEALAND Ltd
Priority date: 2013-04-05
Filing date: 2014-04-04
Publication date: 2017-06-29
Anticipated expiration: 2034-04-04
Also published as: AU2014201934A1

Abstract

Disclosed herein are compositions for euthanising an amphibian and compositions when used for euthanising an amphibian. The compositions comprise eugenol, isoeugenol, or both, one or more surfactant, and one or more macrogol. Also disclosed herein are methods for euthanising an amphibian, the methods comprising contacting the amphibian with a composition comprising eugenol, isoeugenol, or both, one or more surfactant, and one or more macrogol.

Description

Regulation 3.2A

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COMPLETE SPECIFICATION

For the invention entitled: ‘COMPOSITION AND METHOD FOR AMPHIBIAN EUTHANASIA”

Applicant: AQUI-S NEW ZEALAND LIMITED, of 1007 High Street, Lower Hutt 5011,

New Zealand;

Address for service in Australia: A J Park, Level 11, 60 Marcus Clarke Street, Canberra ACT 2601, Australia.

The following statement is a full description of this invention, including the best method of performing known to us: C-' O (N δ'

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COMPOSITION AND METHOD FOR AMPHIBIAN EUTHANASIA FIELD OF THE INVENTION

[0001] The present invention relates to a composition for euthanising an amphibian and to a method of using the composition. More particularly, the invention relates to a composition for euthanising an amphibian, the composition comprising eugenol and/or isoeugenol.

BACKGROUND

[0002] Amphibians are members of the class Amphibia, a group of vertebrates whose living forms include frogs, toads, salamanders, newts and caecilians. Various amphibians, such as the cane toad (Bu/b marinus), are pests. Cane toads, which are native to Central and South America, were introduced to many other countries for the biological control of agricultural pests. Cane toads have become a pest in many of these countries, such as Australia and the United States.

[0003] Amphibians in the early stages of their life cycle (for example, tadpoles) are entirely aquatic and have gills for respiration. Most amphibians lose their gills during metamorphosis and develop lungs. Most importantly, amphibian skin acts as a semipermeable membrane that allows for respiration (cutaneous respiration) and absorption of substances through the skin.

[0004] This skin physiology allows amphibians to be anaesthetized by immersion in an anaesthetic solution, placement in an anaesthetic gas induction chamber, or by anaesthetic preparations applied to the skin. Historically, the anaesthetic of choice for amphibians has been tricaine methanesulfonate (MS-222), which is an isomer of benzocaine. CO2 gassing is the most common euthanasia technique for frogs, however this is controversial. Clove oil, which contains the active constituent eugenol, is an effective anaesthetic and euthanising agent in fish (Ross, L. G. and Ross, B. (2009). Anaesthetic and Sedative Techniques for Aquatic Animals, 3rd ed. Blackwell Publishing, Oxford) and has also been used as an anaesthetic agent in some amphibian species (Mitchell, M.A. (2009). Anesthetic considerations for amphibians. Journal of Exotic Pet Medicine, 18, pp. 40-49).

[0005] The use of clove oil as a euthanising agent for amphibians, specifically cane toads, has been documented, however, the mode of application and the side effects are questionable. Application modes vary from concentrated baths to direct application to the skin. Delayed

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[0006] Accordingly, it is an object of the present invention to go some way to avoiding the above disadvantages; and/or to at least provide the public with a useful choice.

[0007] Other objects of the invention may become apparent from the following description which is given by way of example only.

SUMMARY OF THE INVENTION

[0008] In a first aspect, the present invention provides a composition for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.

[0009] In a second aspect, the present invention provides a composition when used for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol. More specifically, the present invention provides a composition when used for euthanising a post-metamorphic amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.

[0010] In a third aspect, the present invention provides use of: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol; for the manufacture of a composition for euthanising an amphibian. More specifically, the present invention provides use of: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol; for the manufacture of a composition for euthanising a post-metamorphic amphibian.

[0011] The present invention also provides a composition for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant.

[0012] The present invention also provides a composition when used for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant. ο Η Ο (Ν δ' σ^ (Ν m σ^ ο (Ν Ο (Ν [0013] The present invention also provides use of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant; for the manufacture of a composition for euthanising an amphibian.

[0014] The present invention also provides a composition for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[0015] The present invention also provides a composition when used for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[0016] The present invention also provides use of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water; for the manufacture of a composition for euthanising an amphibian.

[0017] The present invention also provides use of a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol; and water for the manufacture of a composition for euthanising an amphibian.

[0018] The present invention also provides a composition for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water.

[0019] The present invention also provides a composition when used for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water.

[0020] The present invention also provides use of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water; for the manufacture of a composition for euthanising an amphibian.

[0021] The present invention also provides use of a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant; and water for the manufacture of a composition for euthanising an amphibian. ο (N S'

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[0022] In a fourth aspect, the present invention provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition of the first or second aspect.

[0023] In a fifth aspect, the present invention provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol. More specifically, the present invention provides a method for euthanising a post-metamorphic amphibian, the method comprising contacting the amphibian with a composition comprising; eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.

[0024] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant.

[0025] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition comprising; eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[0026] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water.

[0027] Although the present invention is broadly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embodiments of which the following description gives examples.

[0028] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

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BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The invention will now be described with reference to the Figures in which: [0030] F igure 1 shows the progression to euthanasia of cane toads (n = 2) after back or belly application of a formulation comprising isoeugenol; [0031] Figure 2 sho ws the progression to euthanasia of various sizes of cane toads (n = 12) after application of a formulation comprising isoeugenol; [0032] Figure 3 sho ws the progression of cane toads (n = 2) to euthanasia after application of a formulation comprising isoeugenol compared with bathing in an isoeugenol bath (25 mg/L); and [0033] Figure 4 shows the progression of small (~50 g) cane toads (n = 12) in their natural environment to euthanasia, compared with pre-caught toads, after application of a formulation comprising isoeugenol.

DETAILED DESCRIPTION

[0034] The term “comprising” as used in this specification means “consisting at least in part of’. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement or claim, all need to be present but other features can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in the same manner.

[0035] Unless othemise specified, all solution concentrations herein are percent weight/weight (% w/w).

[0036] The present invention broadly relates to a composition comprising eugenol and/or isoeugenol for euthanising an amphibian. Advantageously, the composition avoids the side effects observed in, for example, cane toads exposed to clove oil. In some embodiments, the amphibian dies within 5-10 minutes of application of the composition.

[0037] In a first aspect, the present invention provides a composition for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.

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[0038] The present invention also provides a composition for euthanising an amphibian, the composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.

[0039] In a second aspect, the present invention provides a composition when used for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.

[0040] The present invention also provides a composition when used for euthanising an amphibian, the composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.

[0041] In a third aspect, the present invention provides use of: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol; for the manufacture of a composition for euthanising an amphibian.

[0042] In some embodiments, the composition comprises eugenol.

[0043] In some embodiments, the composition comprises eugenol and includes substantially no isoeugenol.

[0044] In some embodiments, the composition comprises isoeugenol.

[0045] In some embodiments, the composition comprises isoeugenol and includes substantially no eugenol.

[0046] In one embodiment, the present invention provides a composition for euthanising an amphibian, the composition comprising eugenol, one or more surfactant, and one or more macrogol.

[0047] In another embodiment, the present invention provides a composition for euthanising an amphibian, the composition comprising isoeugenol, one or more surfactant, and one or more macrogol.

[0048] In another embodiment, the present invention provides a composition for euthanising an amphibian, the composition comprising eugenol, isoeugenol, one or more surfactant, and one or more macrogol. r-- O (N δ'

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[0049] Isoeugenol can exist as two geometric isomers; cis and trans. Isoeugenol is commercially available in forms which contain from about 2-3% by weight to as high as 15% by weight of the cis isomer. The commercially available fonns of isoeugenol are suitable for use in the composition. In some preferred embodiments, the isoeugenol contains about 85-95% by weight of the trans isomer and about 5-15% by weight of the cis isomer. In some preferred embodiments, the isoeugenol contains about 90-92% by weight of the trans isomer and about 8-10% by weight of the cis isomer.

[0050] In some embodiments, however, the isoeugenol is substantially free of the cis isomer, that is with about 2% or less of the cis isomer present. In some embodiments, the trans isomer is greater than about 99% pure, that is with less than about 1% of the cis isomer present, or greater than about 99.5% pure.

[0051] In other embodiments, the isoeugenol is enriched in the cis isomer. In some embodiments, the isoeugenol comprises up to about 80% cis isomer.

[0052] Suitable surfactants are able to be combined with the active agent, that is the eiigenol and/or isoeugenol. Preferably, the surfactant is a liquid at ambient temperature. In some preferred embodiments, the surfactant is a sorbitan ester or a polysorbate.

[0053] In some embodiments, the surfactant is a sorbitan ester.

[0054] In some embodiments, the surfactant is sorbitan oleate, sorbitan sesquioleate, sorbitan isostearate, sorbitan laurate, sorbitan trioleate, or a mixture of any two or more thereof.

[0055] In some embodiments, the surfactant is sorbitan oleate.

[0056] A suitable commercially available sorbitan oleate is SPAN™ 80.

[0057] In some more preferred embodiments, the surfactant is a polysorbate. Polysorbates are nonionic surfactants and emulsifiers derived from PEG-ylated sorbitan esterified with fatty acids.

[0058] In some embodiments, the surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate), polysorbate 60 (polyoxyethylene (20) monostearate), polysorbate 40 ο (Ν S' σ^ (Ν m σ^ ο (Ν Ο (Ν (polyoxyethylene (20) monopalmitate), polysorbate 20 (polyoxyethylene (20) monolaurate), or a mixture of any two or more thereof.

[0059] In some particularly preferred embodiments, the surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). Polysorbate 80 is a viscous, water-soluble yellow liquid.

[0060] A suitable commercially available polysorbate 80 is LIPOSORB™ 0-20. Other commercially available polysorbate 80 include KOLLIPHOR^*''* PS 80, TWEEN™ 80, ALKEST™ TW80, and CANARCEL™ TW80.

[0061] Macrogols are a class of compounds that includes polyethylene glycols (PEGs), also known as polyethylene oxides (PEOs) or polyoxyethylenes (POEs). They are mixtures of condensation polymers of ethylene oxide and water. Those with an average molecular weight of 200-700 are generally liquid at ambient temperature, and are prefeiTed for use in the composition of the invention.

[0062] In some embodiments, the macrogol contains less than about 1.0%, preferably less than about 0.5%, water. In some embodiments, the macrogol is substantially anhydrous, that is containing less than about 0.1% water.

[0063] The macrogol acts as an emulsifier. Without wishing to be bound by theor>% it is thought that the macrogol also aids the diffusion of the eugenol and/or isoeugenol across the amphibian's skin.

[0064] Preferred commercially available liquid macrogols include PEG 200, PEG 300, PEG 400 and PEG 600. In some more preferred embodiments, the macrogol is PEG 400.

[0065] In some embodiments, tlie composition further comprises an antioxidant. The antioxidant slows the breakdown and the discoloration of the composition.

[0066] Suitable antioxidants include the various food grade and/or pharmaceutical antioxidants known in the art. In some preferred embodiments, the antioxidant is selected from tocopherols and fatty acid esters of ascorbic acid. Other suitable antioxidants include, but are not limited to, those comprising monohydroxy- or polyhydroxy-phenols, such as propyl gallate, tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and gossypol.

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[0067] In some more preferred embodiments, the antioxidant is ascorbyl palmitate. Ascorbyl palmitate (also known as [(25')-2-[(2i?)-4,5-dihydroxy-3-oxo-2-furyl]-2-hydroxy-ethyl] hexadecanoate, L-ascorbic acid 6-hexadecanoate, or 6-0-palmitoylascorbic acid) is an ester formed from ascorbic acid and palmitic acid.

[0068] The present invention also provides a composition for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant.

[0069] The present invention also provides a composition for euthanising an amphibian, the composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant.

[0070] The present invention also provides a composition when used for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant.

[0071] The present invention also provides a composition when used for euthanising an amphibian, the composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant.

[0072] The present invention also provides use of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant; for the manufacture of a composition for euthanising an amphibian.

[0073] The proportions of the various components of the composition can vary.

[0074] In some embodiments, the composition comprises: about 1-50% eugenol and/or isoeugenol; about 0.5-50% surfactant; about 25-95% macrogol; and about 0-1% antioxidant.

[0075] In some preferred embodiments, the composition comprises: about 5-25% eugenol and/or isoeugenol; about 0.5-10% surfactant; ο Η Ο (Ν δ' σ^ (Ν m σ^ ο (Ν Ο (Ν 10 about 65-94.5% macrogol; and about 0-1% antioxidant.

[0076] In some more preferred embodiments, the composition comprises: about 5-15% eugenol and/or isoeugenol; about 0.5-5% surfactant; about 85-94.5% macrogol; and about 0-1% antioxidant.

[0077] In some preferred embodiments, the composition comprises: about 1-50% eugenol and/or isoeugenol; about 0.5-50% surfactant; about 25-95% macrogol; and about 0-0.1% antioxidant.

[0078] In some particularly preferred embodiments, the composition comprises: about 10% eugenol and/or isoeugenol; about 1% surfactant; about 88.95% macrogol; and about 0.05% antioxidant.

[0079] In some preferred embodiments, the composition comprises: about 1-50% eugenol and/or isoeugenol; about 0.5-50% polysorbate; about 25-95% polyethylene glycol; and about 0-1% ascorbyl palmitate.

[0080] In some more preferred embodiments, the composition comprises: about 5-25% eugenol and/or isoeugenol; about 0.5-10% polysorbate; about 65-94.5% polyethylene glycol; and about 0-1% ascorbyl palmitate.

[0081] In some even more preferred embodiments, the composition comprises: about 5-15% eugenol and/or isoeugenol; about 0.5-5% polysorbate;

r- ο (N

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m σ^ o (N O (N 11 about 85-94.5% polyethylene glycol; and about 0-1% ascorbyl palmitate.

[0082] In some more preferred embodiments, the composition comprises: about 1-50% eugenol and/or isoeugenol; about 0.5-50% polysorbate; about 25-95% polyethylene glycol; and about 0-0.1% ascorbyl palmitate.

[0083] In some particularly preferred embodiments, the composition comprises: about 10% eugenol and/or isoeugenol; about 1% polysorbate; about 88.95% polyethylene glycol; and about 0.05% ascorbyl palmitate.

[0084] Advantageously, in those embodiments in which the composition comprises eugenol, the composition is effective for euthanising amphibians despite including a lower concentration of eugenol than clove oil, which may comprise about 70-98%, more typically about 75-85%, eugenol.

[0085] The use of a composition with a lower concentration of eugenol than clove oil reduces the risk of gastric prolapse, or other adverse reaction when the composition is applied to tlie amphibian. It also increases the possibility of recovery in the event of accidental contact of an amphibian with the composition.

[0086] In some particularly preferred embodiments, the composition comprises eugenol, polysorbate 80, polyethylene glycol and ascorbyl palmitate. In some of these embodiments, the composition consists essentially of eugenol, polysorbate 80, polyethylene glycol and ascorbyl palmitate. In a particularly preferred embodiment, the composition consists essentially of eugenol (10%), polysorbate 80 (1%), polyethylene glycol (PEG 400; 88.95%) and ascorbyl palmitate (0.05%). In another particularly preferred embodiment, the composition consists essentially of eugenol (5%), polysorbate 80 (0.5%), polyethylene glycol (PEG 400; 94.45%) and ascorbyl pahnitate (0.05%). In another particularly preferred embodiment, the composition consists essentially of eugenol (25%), polysorbate 80 (2.5%), polyethylene glycol (PEG 400; 72.45%) and ascorbyl palmitate (0.05%).

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m σ^ o (N O (N 12 [0087] In some other particularly preferred embodiments, the composition comprises isoeugenol, polysorbate 80, polyethylene glycol and ascorbyl palmitate. In some of these embodiments, the composition consists essentially of isoeugenol, polysorbate 80, polyethylene glycol and ascorbyl palmitate. In a particularly preferred embodiment, the composition consists essentially of isoeugenol (10%), polysorbate 80 (1%), polyethylene glycol (PEG 400; 88.95%) and ascorbyl palmitate (0.05%). In another particularly preferred embodiment, the composition consists essentially of isoeugenol (5%), polysorbate 80 (0.5%), polyethylene glycol (PEG 400; 94.45%) and ascorbyl palmitate (0.05%). In another particularly preferred embodiment, the composition consists essentially of isoeugenol (25%), polysorbate 80 (2.5%), polyethylene glycol (PEG 400; 72.45%) and ascorbyl palmitate (0.05%).

[0088] In some embodiments, the composition further comprises methyl salicylate, ethyl salicylate, or both. Without wishing to be bound by theory, it is thought that the inclusion of methyl salicylate and/or ethyl salicylate in the composition increases the heart rate of the amphibian and improves the efficacy of the composition.

[0089] In some embodiments, the composition comprises about 1-25% methyl salicylate, ethyl salicylate, or both. In some embodiments, the composition comprises about 1-10% methyl salicylate, ethyl salicylate, or both. In some embodiments, the composition comprises about 1-5% methyl salicylate, ethyl salicylate, or both.

[0090] The composition may further comprise one or more other component. Other components may be selected from, but are not limited to, diluents, thickeners, stabilisers, amphibian attractants, and repellents for non-target species.

[0091] In some embodiments, the composition further comprises a diluent. In some preferred embodiments, the diluent is water. In some embodiments, the invention provides an aqueous composition comprising about 5-95 v/v% (percent volume/volume) of a composition as described above and further comprising about 95-5 v/v% water. In some embodiments, the invention provides an aqueous composition comprising about 9-95 v/v% of a composition as described above and further comprising about 91-5 v/v% water. In some embodiments, the aqueous composition comprises 1 volume of a composition as described above and about 0.05-10 volumes of water.

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σ^ o (N O (N 13 [0092] Accordingly, the present invention also provides a composition for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[0093] The present invention also provides a composition for euthanising an amphibian, the composition consisting essentially of; eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[0094] The present invention also provides a composition when used for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[0095] The present invention also provides a composition when used for euthanising an amphibian, the composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[0096] The present invention also provides use of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water; for the manufacture of a composition for euthanising an amphibian.

[0097] The present invention also provides use of a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol; and water for the manufacture of a composition for euthanising an amphibian.

[0098] The present invention also provides use of a composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol; and water for the manufacture of a composition for euthanising an amphibian.

[0099] The present invention also provides a composition for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water.

[00100] The present invention also provides a composition for euthanising an amphibian, the composition consisting essentially of; eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water. Ο (Ν S' σ^ (Ν m σ^ ο (Ν Ο (Ν 14 [00101J The present invention also provides a composition when used for euthanising an amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water.

[00102] The present invention also provides a composition when used for euthanising an amphibian, the composition consisting essentially of; eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water.

[00103J The present invention also provides use of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water; for the manufacture of a composition for euthanising an amphibian.

[00104] The present invention also provides use of a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant; and water for the manufacture of a composition for euthanising an amphibian.

[00105] The present invention also provides use of a composition consisting essentially of; eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant; and water for the manufacture of a composition for euthanising an amphibian.

[00106] In some particularly preferred embodiments, all of the components of the composition are non-hazardous, that is safe for human consumption. In particular, the active compounds eugenol and isoeugenol are GRAS (generally recognised as safe) compounds, as are methyl salicylate and ethyl salicylate, and the excipients are FDA (US Food and Drug Administration) and EPA (US Environmental Protection Agency) approved inert ingredients. As such, the composition is suitable for household use and its use does not require specialist equipment or training.

[00107] The composition is particularly useful for euthanising cane toads. The composition may be used for euthanising other amphibians, for example, other pest species such as the common coqui or coqui (Eleutherodactylus coqui) and the American hullfrog {Rana catesbeiana).

[00108] The composition of the invention is also suitable for amphibian euthanasia, for example in a veterinary or curatorial setting.

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σ^ H O (N H O (N 15 [00109] The composition of the invention is suitable for direct application to an amphibian without requiring dilution or other further formulation steps. The invention is not, however, limited thereto and, in some embodiments, the composition is mixed with a suitable diluent prior to application. For example, in some embodiments, the composition is mixed with water prior to application.

[00110] In some embodiments, the composition comprises a spray formulation.

[00111] Devices suitable for delivering a solution in the form of a spray are well known to those skilled in the art. Suitable devices include mechanical sprayers that pump the solution from a reservoir, and aerosol devices that utilise compressed propellant gases to generate a spray through a nozzle.

[00112] Accordingly, some embodiments of the invention provide a mechanical sprayer having a reservoir loaded with a composition of the invention.

[00113] Some embodiments of the invention provide an aerosol device comprising a solution or suspension comprising one or more propellants and a composition of the invention.

[00114] In some embodiments, the mechanical sprayer or aerosol device delivers a stream or jet spray of the composition. Advantageously, a stream spray is more accurate at a longer range, and is less affected by wind, than a mist spray.

[00115] The invention also contemplates embodiments in which a mechanical sprayer or aerosol device is incorporated into a device capable of detecting an amphibian and spraying the composition onto the amphibian.

[00116] In a fourth aspect, the present invention provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition of the first or second aspect.

[00117] In a fifth aspect, the present invention provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol. Ο Η Ο (Ν δ' σ^ (Ν m σ^ ο (Ν Ο (Ν 16 [00118J The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.

[00119] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant.

[00120] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and one or more antioxidant.

[00121] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[00122] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; and water.

[00123] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water.

[00124] The present invention also provides a method for euthanising an amphibian, the method comprising contacting the amphibian with a composition consisting essentially of: eugenol, isoeugenol, or both; one or more surfactant; one or more macrogol; one or more antioxidant; and water.

[00125] Suitable compositions for use in the method of the fifth aspect include those described above in relation to the first or second aspect. ο Η Ο (Ν δ' σ^ (Ν σ^ Η Ο (Ν Η Ο (Ν 17 [00126J In a preferred embodiment, the amphibian is contacted with the composition by topical application of the composition. In some more preferred methods, a person applying the composition is not required to physically contact the amphibian.

[00127] In some embodiments, the composition is suitable for direction application to the amphibian without requiring dilution or other formulation steps. The invention is not, however, limited thereto and, in some other embodiments, the composition is mixed with a suitable diluent prior to application. For example, in some embodiments, the composition is mixed with water prior to application.

[00128] The composition may be topically applied to the amphibian by, for example, brushing, wiping, dripping or spraying the composition onto the dorsum of the amphibian.

[00129] In some preferred embodiments, the composition is topically applied to the amphibian by spraying the composition onto the dorsum of the amphibian.

[00130] The composition may also be topically applied to the amphibian by at least partially immersing the amphibian in the composition. For example, in some embodiments, the composition may be held in a reservoir in a trap. The invention is not, however, limited thereto.

[00131] Advantageously, in the event of accidental contact of an amphibian with the composition, the amphibian may be recovered by washing the composition from the amphibian prior to euthanasia. The amphibian is generally washed with copious amounts of clean water to remove the composition. In some embodiments, the amphibian is at least partially immersed in clean water following washing.

[00132] Conveniently, the method of the invention euthanises the amphibian without requiring additional treatment of the amphibian, for example freezing following sedation.

[00133] The composition of the invention provides for a rapid uptake of eugenol and/or isoeugenol. In some embodiments, the amphibian is deeply sedated within about 2-5 minutes of being contacted with the composition. In some preferred embodiments, the amphibian dies within about 10-20 minutes of being contacted with the composition. In some more preferred embodiments, the amphibian dies within about 10-15 minutes of being contacted with the composition. ο Η Ο (Ν σ^ (Ν m σ^ ο (Ν Ο (Ν 18 [00134J Rapid uptake of the eugenol and/or isoeugenol is advantageous because it results in rapid cessation of movement of the amphibian. The amphibian will not, therefore, move far, if at all, from the point of contact with the composition. This simplifies retrieval and disposal of the euthanised amphibian.

[00135j The amount of composition applied to the amphibian can vary. In some embodiments, about 0.01-0.1 ml of composition is applied per gram of amphibian body weight. In some embodiments, the composition is applied to provide a dose of eugenol and/or isoeugenol of about 0.001-0.01 ml per gram of amphibian body weight. In some embodiments, about 0.01-0.1 g of composition is applied per gram of amphibian body weight. In some embodiments, the composition is applied to provide a dose of eugenol and/or isoeugenol of about 0.001-0.01 g per gram of amphibian body weight.

[00136] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[00137] The following non-limiting examples are provided to illustrate the present invention and in no way limit the scope thereof

EXAMPLES

Example 1 [00138] The trial formulation was a water dispersible composition comprising 10% eugenol as the active ingredient. The composition further comprised polysorbate 80 (1%), polyethylene glycol (PEG 400; 88.95%) and ascorbyl palmitate (0.05%).

[00139] Target Species; Cane toad {Bufo marinus). The cane toad is very large; the females are significantly longer than males, reaching an average length of 10-15 cm. The biggest toad recorded in Australia measured 24 cm long and weighed 1.3 kg. The juvenile cane toad is much smaller than the adult cane toad at 5-10 cm long. The tadpoles range from 10-25 mm in length. r- ο (N δ'

σ^ (N

m σ^ o (N O (N 19 [00140J Application; 5 ml applied directly to the back of the toad. Toad left undisturbed for 4 minutes in a bucket.

[00141] Observations: When applied, the toad made no attempt to escape, suggesting there was no adverse effect from the composition being applied. Over the 4 minutes the toad did not move. When handled after 5 minutes exposure, the toad responded with an escape response. After 6 minutes the toad was dead. An attempt was made to recover the toad after 9 minutes by rinsing the composition off the toad. However, no recovery was achieved, confirming the toad had been effectively euthanised. No other observations were noted.

Example 2 [00142] Three formulations were tested comprising different eugenol concentrations: - 5% eugenol, polysorbate 80 (0.5%), polyethylene glycol (PEG 400; 94.45%) and ascorbyl palmitate (0.05%); - 10% eugenol, polysorbate 80 (1%), polyethylene glycol (PEG 400; 88.95%) and ascorbyl palmitate (0.05%); and - 25% eugenol, polysorbate 80 (2.5%), polyethylene glycol (PEG 400; 72.45%) and ascorbyl palmitate (0.05%).

[00143] Target Species: Cane toad {Bufo marinus).

[00144] Application: 3 ml applied directly to the back of the toad. Toad left undisturbed in a bucket.

[00145] Observations: For all three formulations there was no attempt to escape when the composition was being applied. There was some activity from the toad over the next 5 minutes however this was minor. After 6 min 24 sec the toad exposed to the formulation comprising 5% eugenol was dead. It took 7 min 24 sec for the toad exposed to 10 % eugenol to die, and 8 min 39 sec for the toad exposed to 25% eugenol. No other observations were noted.

r- ο (N

σ^ (N

σ^ o (N O (N 20

Example 3

Trial I - Pre-captured toads [00146] Trial I was conducted on pre-caught toads. Toads of varying size (50-200 g) were cauglit the previous night from four local locations.

[00147] Several small trials were conducted on these animals to determine the following: - initial toad reactions to being sprayed with the trial formulation, which comprised 5% isoeugenol, polysorbate 80 (0.5%), polyethylene glycol (PEG 400; 94.4%) and ascorbyl palmitate (0.1%); - stages of sedation in cane toads; - best application method - back or belly; - time to euthanasia versus toad size; and - difference between the trial formulation and an isoeugenol bath (25 mg/L).

Trial II - Natural environment [00148] Trial II examined the reaction of toads to the trial formulation in their natural environment. This trial was done just after sundown. Small toads of approximately 50-100 g were most common in this area. The trial formulation was applied to the animal's back without otherwise disturbing the toad. The initial reaction was noted and the time to euthanasia recorded.

Results [00149] In all the animals tested, none showed adverse reaction to the trial fonnulation being applied. This was demonstrated by the toad remaining still and not attempting to escape the area after the initial application. This demonstrates that there is no irritation to the animals and that there is no undue stress or suffering being caused by the trial formulation.

Stages of sedation [00150] Overall, pre-caught and wild cane toads from trials I and 11 appeared to go through several key identifiable stages before they became euthanised. These stages were clear and well defined and could be used to identify the level of sedation in the animal (Table 1). 21 r- ο (N δ' [00151J In cane toads the following four key stages were identified: Table 1. Progress of sedation to euthanasia in cane toads.

σ^ (N

m σ^ o (N O (N

Stage Descriptor Toad behavioural response during each stage Initial behaviour Final behaviour 1 Light sedation No visual change in behaviour is observed Loss of reaction to non-invasive external stimuli. No response to a hand or an object being waved in front of the animal II Deep sedation Hopping is replaced by crawling when exposed to invasive external stimuli No reaction to external stimuli but when an animals is placed on its back it is still able to reright itself III Deep anaesthesia Complete loss of equilibrium; breathing action significantly slowed Breathing action stopped but some eye response when exposed to invasive external stimuli (light touching) IV Euthanasia Complete loss of eye reaction; air from body cavity exhaled resulting in toad of 1/3 the size N/A

Application method [00152] Application of the trial fonnulation directly to the back of the animal proved to be significantly more effective in euthanising the animal than application on the belly (Figure 1). Toads sprayed with the trial formulation on their back progressed to Stage IV (euthanasia) within 15 minutes. Toads where belly application was made were slower, progressing to Stage IV after 18 minutes. This suggests that there is better transportation of the active ingredient (isoeugenol) across the backs of toads and it is, therefore, the preferred method of application.

Dose response [00153] Size variation was seen to have a significant impact on the time and pattern of progression to euthanasia (Figure 2). It was observed that small toads (~50 g) were slow to progress to stage III sedation (11 minutes). Medium (~100 g) and large (-150-200 g) animals, however, were significantly faster, progressing to stage III sedation within 8 minutes. Small animals then progressed to euthanasia (stage IV) within another 2 minutes; or

ο (N

σ^ (N

m σ^ o (N O (N 22 13 minutes after initial application. Medium sized animals and large animals progressed slower, dying 14 and 15 minutes after application, respectively.

The trial formulation versus isoeugenol bath [00154] There was a significant difference in the progression to euthanasia of toads treated with the trial formulation compared to those placed in a 25 mg/L isoeugenol sedation bath (Figure 3). The isoeugenol sedation bath was prepared by appropriate dilution of AQUl-S® (50% v/v isoeugenol), a commercially-available fish anaesthetic developed by AQUl-S New Zealand Limited. The toads sprayed with the trial formulation died after 15 minutes and followed the general progression to euthanasia as seen above. The toad in the isoeugenol bath, however, had not progressed past Stage I sedation after 15 minutes and was still showing reaction to external stimuli and an escape response after 30 minutes. This may have been due to the low concentration of isoeugenol used in the bath or the fact that the toad only had its belly exposed to the isoeugenol solution facilitating the poor uptake of isoeugenol.

Reaction to the trial formulation in the wild [00155J It was observed that wild cane toads had no initial adverse reactions to being sprayed by the trial formulation in their natural environment. Upon application of the trial formulation, the toads had no desire to move or flee from the area of application. Of the 10 toads sprayed in the wild, none fled the area until they were manually disturbed by poking. These results support the findings of the first trial.

Dose response [00156J Small toads (~50 g), to whom the trial formulation was applied in their natural environment, progressed to Stage IV (euthanasia) after 14 minutes (Figure 4). This was 1 minute slower than similar sized pre-caught toads, as demonstrated above. This suggests that possible stress in the toads (demonstrated in pre-caught animals) has little effect on the time and progression to euthanasia.

[00157J This study has demonstrated the effectiveness of the isoeugenol-based trial formulation as a toad euthanising agent. It w-as observed that size, application method and application type (the trial formulation compared with isoeugenol bath) had a significant impact on the time and progression profile to euthanasia in toads. Whether the toads were

ο (N

σ^ (N

σ^ o (N O (N 23 pre-handled (that is, pre-caught), and possibly stressed, or sprayed in their natural environment had little impact on the rate and progression to euthanasia.

[00158] These results demonstrated that toads can be euthanised with an isoeugenol-based formulation in a humane manner with minimal handling.

INDUSTRIAL APPLICATION

[00159] It will be appreciated from the discussion above that the present invention provides a composition for euthanising an amphibian and a method of using the composition for euthanising an amphibian.

[00160] The composition and method are particularly useful for euthanising pest amphibians, such as cane toads {Bufo marinus). Advantageously, the amphibian may be euthanised by, for example, spraying the liquid composition onto the dorsum of the amphibian, which method does not require any physical contact with the amphibian.

[00161] Those persons skilled in the art will understand that the above description is provided by way of illustration only and that the invention is not limited thereto.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A composition when used for euthanising a post-metamorphic amphibian, the composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.
2. Use of: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol; for the manufacture of a composition for euthanising a post-metamorphic amphibian.
3. A method for euthanising a post-metamorphic amphibian, the method comprising contacting the amphibian with a composition comprising: eugenol, isoeugenol, or both; one or more surfactant; and one or more macrogol.
4. A composition as claimed in claim 1, use as claimed in claim 2 or method as claimed in claim 3, wherein the composition further comprises one or more antioxidant.
5. A composition, use or method as claimed in any one of claims 1 to 4, wherein the composition comprises: about 1-50% eugenol and/or isoeugenol; about 0.5-50% surfactant; about 25-95% macrogol; and about 0-1% antioxidant.
6. A composition, use or method as claimed in any one of claim 1 to 4, wherein the composition comprises: about 5-25% eugenol and/or isoeugenol; about 0.5-10% surfactant; about 65-94.5% macrogol; and about 0-1% antioxidant.
7. A composition, use or method as claimed in any one of claim 1 to 4, wherein the composition comprises: about 5-15% eugenol and/or isoeugenol; about 0.5-5% surfactant; about 85-94.5% macrogol; and about 0-1% antioxidant.
8. A composition, use or method as claimed in any one of claim 1 to 4, wherein the composition comprises: about 1-50% eugenol and/or isoeugenol; about 0.5-50% surfactant; about 25-95% macrogol; and about 0-0.1% antioxidant.
9. A composition, use or method as claimed in any one of claim 1 to 4, wherein the composition comprises: about 10% eugenol and/or isoeugenol; about 1% surfactant; about 88.95% macrogol; and about 0.05% antioxidant.
10. A composition, use or method as claimed in any one of claims 1 to 9, wherein the composition comprises eugenol.
11. A composition, use or method as claimed in any one of claims 1 to 10, wherein the composition comprises eugenol and includes substantially no isoeugenol.
12. A composition, use or method as claimed in any one of claims 1 to 11, wherein the surfactant is a sorbitan ester or a polysorbate.
13. A composition, use or method as claimed in any one of claims 1 to 12, wherein the surfactant is a polysorbate.
14. A composition, use or method as claimed in any one of claims 1 to 13, wherein the surfactant is polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, or a mixture of any two or more thereof.
15. A composition, use or method as claimed in any one of claims 1 to 14, wherein the surfactant is polysorbate 80.
16. A composition, use or method as claimed in any one of claims 1 to 15, wherein the macrogol is a polyethylene glycol (PEG).
17. A composition, use or method as claimed in any one of claims 1 to 16, wherein the macrogol is PEG 200, PEG 300, PEG 400, or PEG 600, or a mixture of any two or more thereof.
18. A composition, use or method as claimed in any one of claims 1 to 17, wherein the macrogol is PEG 400.
19. A composition, use or method as claimed in any one of claims 1 to 18, wherein the composition is topically applied to the amphibian.
20. A composition, use or method as claimed in any one of claims 1 to 19, wherein the composition is topically applied to the dorsum of the amphibian.
21. A composition, use or method as claimed in any one of claims 1 to 20, wherein the composition is a spray formulation.
22. A composition, use or method as claimed in any one of claims 1 to 19, wherein the amphibian is deeply sedated within about 2-5 minutes of being contacted with the composition.
23. A composition, use or method as claimed in any one of claims 1 to 22, wherein the amphibian dies within about 10-20 minutes of being contacted with the composition.
24. A composition, use or method as claimed in any one of claims 1 to 23, wherein the amphibian dies within about 10-15 minutes of being contacted with the composition.
25. A composition, use or method as claimed in any one of claims 19 to 21, wherein about 0.01-0.1 ml of composition is applied per gram of amphibian body weight.
26. A composition, use or method as claimed in any one of claims 19 to 21, wherein about 0.01-0.1 g of composition is applied per gram of amphibian body weight.
27. A composition, use or method as claimed in any one of claims 19 to 21, wherein the composition is applied to provide a dose of eugenol and/or isoeugenol of about 0.001-0.01 ml per gram of amphibian body weight.
28. A composition, use or method as claimed in any one of claims 19 to 21, wherein the composition is applied to provide a dose of eugenol and/or isoeugenol of about 0.001-0.01 g per gram of amphibian body weight.
29. A composition, use or method as claimed in any one of claims 1 to 28, wherein the amphibian is a cane toad (Bufo marinus).