AU2013206224A1 - Chemical Attractant and Use Thereof - Google Patents

Abstract

H:\sxf\Intrwovn\NRPortb\DCC\SXF\5228869I.doc-7/06/2013 The present invention relates to methods of trapping tadpoles from the family Bufonidae and thereby controlling populations of toads from this family. Compositions comprising at least one chemical tadpole attractant and sustained release compositions and devices comprising such attractant compositions are also described. The chemical tadpole attractants are derived from the eggs or parotoid secretions of toads of the Bufonidae family or comprise bufagenin compounds or the 3-0-esters.

Description

H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 CHEMICAL ATTRACTANT AND USE THEREOF Field of the Invention The present invention relates to methods of trapping tadpoles from the family Bufonidae 5 and thereby controlling the population of toads from this family. Compositions comprising at least one chemical tadpole attractant and sustained release compositions and devices comprising such attractant compositions are also described. Background of the Invention 10 The cane toad Rhinella marina, formerly known as Bufo marinus, is an invasive pest that was introduced to multiple locations around the world as an ultimately unsuccessful biocontrol agent for beetle pests of sugar cane. In several locations, including Australia, Fiji and Hawaii, the species has had a significant ecological impact on native animal populations. The cane toad, and many other members of the family Bufonidae, are 15 renowned for their ability to produce and deploy cardiotoxic steroids (bufadienolides) as a form of chemical defence. Bufadienolides are antagonists of Na*/K*-ATPase in much the same way as the plant derived cardenolides (such as digitalis) and ingestion can lead to cardiac arrest and death. The prospect of fatal encounters with cane toads is enhanced by the presence of specialised parotoid glands that secrete high concentrations of 20 bufadienolides in response to predatory attack. In Australia, the cane toad is a threat to native predator species such as fresh water crocodiles, marsupials, snakes and lizards, which are highly vulnerable to cane toad poisoning. Similar concerns have been expressed in other countries confronted by this invasive pest animal. 25 Since its release in Australia in 1935, the cane toad has advanced south along the eastern seaboard from Queensland into New South Wales, and west through the Northern Territory into Western Australia - colonising >1 million km2 and seriously impacting native predator populations. To date, cane toad control has been limited to local and short-term techniques such as hand collection, barriers and trapping. Unfortunately, none of the 30 existing cane toad control strategies has halted or even seriously diminished the impact of the cane toad invasion. There remains an urgent and compelling need for an effective H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -2 solution that can be deployed at minimal cost, is environmentally safe, species specific, and acceptable to communities and the legislature. More importantly, such a solution must be deployable on a nation-wide scale, and must offer the realistic hope of dramatically diminishing cane toad numbers and/or generational recruitment. 5 Summary of the Invention The present invention is predicated in part on the discovery that cane toad eggs release chemicals responsible for attracting the attention of cannibalistic cane toad tadpoles and that equivalent chemical attractants can be found in the parotoid secretions of adult cane 10 toads. In one aspect of the invention there is provided a method of controlling a population of toads from the family Bufonidae comprising: (i) exposing tadpoles from the family Bufonidae to a composition comprising or consisting 15 of one of the following: (a) an extract obtained from the eggs of a toad of the family Bufonidae; (b) a parotoid secretion of a toad of the family Bufonidae; and (c) an extract obtained from the parotoid secretion of a toad of the family Bufonidae; and 20 (ii) trapping the tadpoles. In another aspect of the invention, there is provided a method of controlling a population of toads from the family Bufonidae comprising: (i) exposing tadpoles from the family Bufonidae to a composition comprising at least one 25 bufagenin compound of formula (I): H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -3 0 0 R6 R7 CH3 R3 H R, R2 R5- 0#0 R4(I) wherein

R

1 is -OH and R 2 is hydrogen or -OH or R 1 and R 2 taken together form -0-; 5 R 3 is methyl or -CHO;

R

4 is hydrogen or -OH;

R

5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH; 10 R 9 is -CH 3 or -CH 2 OH; t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10; and 15 (ii) trapping the tadpoles. In some embodiments of these aspects, the composition is derived from toad eggs of the family Bufonidae. In other embodiments, the composition is derived from a secretion from the parotoid gland of the family Bufonidae. In some embodiments, the composition 20 comprises or consists of a secretion from the parotoid gland of the family Bufonidae. In particular embodiments, the toad of the family Bufonidae is a toad of the species H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -4 Rhinella marina. In yet another aspect of the invention there is provided a method of controlling a population of tadpoles of the family Bufonidae in a body of water comprising: 5 a) providing a trapping device to a body of water where the body of water hosts a population of tadpoles from the family Bufonidae; b) baiting the trapping device with a tadpole attractant composition comprising or consisting of one of the following: (i) an extract obtained from the eggs of a toad of the family Bufonidae; 10 (ii) a parotoid secretion of a toad of the family Bufonidae; and (iii) an extract obtained from the parotoid secretion of a toad of the family Bufonidae; and c) harvesting the tadpoles from the trapping device. 15 In another aspect of the invention there is provided a method of controlling a population of tadpoles of the family Bufonidae in a body of water comprising: a) providing a trapping device to a body of water where the body of water hosts a population of tadpoles of the family Bufonidae; 20 b) baiting the trapping device with a tadpole attractant composition comprising at least one bufagenin compound of formula (I): 0 0 R6 R7 CH3 R3 H R, R2 R5--O R4() H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -5 wherein

R

1 is -OH and R 2 is hydrogen or -OH or R 1 and R 2 taken together form -0-;

R

3 is methyl or -CHO;

R

4 is hydrogen or -OH; 5 R 5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; t is an integer from 3 to 18; 10 u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10; and c) harvesting the tadpoles from the trapping device. 15 In some embodiments of these aspects, the tadpole from the family Bufonidae is a cane toad tadpole of the species Rhinella marina. In some embodiments, the composition is a sustained release composition or is applied to a device that provides sustained release. 20 In a further aspect of the invention there is provided a use of a composition comprising or consisting of one of the following: a) an extract obtained from the eggs of a toad of the family Bufonidae; b) a parotoid secretion of a toad of the family Bufonidae; and 25 c) an extract obtained from the parotoid secretion of a toad of the family Bufonidae; as an attractant for tadpoles of the family Bufonidae. In yet another aspect of the invention there is provided a use of a composition comprising 30 at least one bufagenin compound of formula (I): H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -6 0 0 R6 R7 CH3 R3 H R, R2 R5--O R4(I) wherein

R

1 is -OH and R 2 is hydrogen of -OH or R 1 and R 2 taken together form -0-;

R

3 is methyl or -CHO; 5 R4 is hydrogen or -OH;

R

5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; 10 t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10; as an attractant for tadpoles of the family Bufonidae. 15 In particular embodiments of these aspects, the tadpole of the family Bufonidae is from the species Rhinella marina. In a further aspect of the invention there is provided a sustained release device comprising: 20 a) a water insoluble matrix; b) a composition comprising or consisting of one of the following: i) an extract obtained from the eggs of a toad of the family Bufonidae; H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -7 ii) a parotoid secretion of a toad of the family Bufonidae; and iii) an extract obtained from the parotoid secretion of a toad of the family Bufonidae. 5 In yet a further aspect of the invention there is provided a sustained release device comprising: a) a water insoluble matrix; and b) a composition comprising at least one bufagenin compound of formula (I): 0 0 R6 R7 CH3 R3 H R, R2 R5-O R4() 10 wherein R1 is -OH and R2 is hydrogen of -OH or R1 and R2 taken together form -O-; R3 is methyl or -CHO; R4 is hydrogen or -OH;

R

5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ; 15 R 6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; t is an integer from 3 to 18; u is an integer from 1 to 10; 20 v is an integer from 1 to 4; and w is an integer from 1 to 10. In some embodiments of these aspects, the water insoluble matrix is a porous organic H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 substrate such as a sponge. In other embodiments, the water insoluble matrix is a porous inorganic substrate such as a glass frit or porous ceramic matrix. In yet a further aspect of the invention there is provided a composition comprising at least 5 one bufagenin compound of formula (I): 0 0 R6 R7 H,3 Rs wherein

R

1 is -OH and R 2 is hydrogen -OH or R 1 and R 2 taken together form -0-;

R

3 is methyl or -CHO; 10 R 4 is hydrogen or -OH; Rs is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ; Rs and R 7 are both hydrogen or together form =0;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; 15 t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10; and wherein the composition is free of hallucinogenic alkaloids. 20 In some embodiments, the composition is an extract obtained from the eggs of a toad of the family Bufonidae. In other embodiments, the composition is an extract obtained from the parotoid secretion of an adult or juvenile toad of the family Bufonidae.

H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -9 In some embodiments, the composition comprises at least one of the following compounds marinobufagenin (1), telocinobufagin (2) and bufalin (3). 0 0 0 0 CH, CH, CH3 H C HICH, H CJ O H OH0 HO HO OH OH 0 0 (3) CH3 H H OH HOO C H (3) 5 In other embodiments, the composition comprises at least one 3-0-ester of a bufagenin compound of formula (I): H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 10 0 0 R6 R7 CH3 R3 H R, R2 R5-O R4() wherein R1 is -OH and R2 is hydrogen -OH or R1 and R2 taken together form -O-; 5 R3 is methyl or -CHO; R4 is hydrogen or -OH;

R

5 is -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH; 10 R 9 is -CH 3 or -CH 2 OH; t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10. 15 In yet another aspect of the invention there is provided a kit comprising: i) a sustained release device of the invention; and ii) an attachment that may be fitted to a container, wherein the attachment is fitted with a funnel shaped cylinder. 20 H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 11 Detailed Description of the Invention A singular forms "a", "an" and "the" include plural aspects unless the context clearly indicates otherwise. 5 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 10 In one aspect of the invention there is provided a method of controlling a population of toads from the family Bufonidae comprising: (i) exposing tadpoles from the family Bufonidae to a composition comprising or consisting of one of the following: (a) an extract obtained from the eggs of a toad of the family Bufonidae; 15 (b) a parotoid secretion of a toad of the family Bufonidae; and (c) an extract obtained from the parotoid secretion of a toad of the family Bufonidae; and (ii) trapping the tadpoles. 20 In another aspect of the invention, there is provided a method of controlling a population of toads from the family Bufonidae comprising: (i) exposing tadpoles from the family Bufonidae to a composition comprising at least one bufagenin compound of formula (I): H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 12 0 R6 R7 CH3 R3 H H R1 R R5 -0 R4(I) wherein

R

1 is -OH and R 2 is hydrogen or -OH or R 1 and R 2 taken together form -0-;

R

3 is methyl or -CHO; 5 R4 is hydrogen or -OH;

R

5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; 10 t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10; and (ii) trapping the tadpoles. 15 In some embodiments of these aspects, the composition is derived from toad eggs of the family Bufonidae. In these embodiments, the composition may comprise crushed toad eggs. In other embodiments, the composition comprises an extract from toad eggs. 20 However, toad eggs are only seasonally available and are difficult to collect in sufficient quantities for preparation of the extract composition. Advantageously, the inventors have discovered that the secretion from the parotoid gland of the juvenile or adult toad also H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 13 comprises compounds that are tadpole attractants. In particular embodiments, these attractant compounds are bufagenin compounds. Therefore, in some embodiments of the invention, the composition is a composition comprising or consisting of the parotoid secretion of a toad from the family Bufonidae. 5 It is known that the parotoid secretion of a toad of the family Bufonidae and particularly Rhinella marina contains bufadienolide compounds featuring a 3-hydroxy functionaility (commonly known as bufagenins), and bufadienolide compounds featuring a 3-0 suberoylarginine ester (commonly known as bufotoxins), and hallucinogenic alkaloids, 10 (such as 5-methoxy-N,N-dimethyltryptamine and bufotenine). As used herein, bufagenins refer to compounds of formula (I) in which R 5 is H. As used herein, bufotoxins refer to compounds of formula (I) in which R 5 is: NH 0 H

H

2 N N H 15

CO

2 H 0 As used herein, 5-methoxy-N,N-dimethyltryptamine is a compound having the following formula: MeO N-CH3 H3C N H 20 As used herein, bufotenine is a compound having the following formula: HO N _-CH3 | CH3

H

H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 14 To reduce the risk of introducing hallucinogenic alkaloids into the bodies of water in which the tadpoles are being trapped, the attractant components of the parotoid secretion such as bufagenin compounds can be isolated readily by extraction. Therefore in some embodiments, the composition comprises or consists of an extract derived from the 5 parotoid secretion of the toad. In particular embodiments, the extract composition comprises at least one bufagenin compound of formula (I), especially a composition that is free of 5-methoxy-N,N-dimethyltryptamine and/or bufotenine. In some embodiments, the extract derived from the parotoid secretion is prepared by 10 collecting the parotoid secretion by applying pressure to the parotoid gland of a juvenile or adult toad of the family Bufonidae and collecting the milky white secretion. The secretion from a number of toads can be combined. The secretion may then be added to water at ambient temperature for at least 30 minutes immediately after collection. This step facilitates the hydrolysis of 3-0-suberoylarginine esters of bufotoxins to provide the 15 bufagenin compounds of formula (I) delivering a product rich in bufagenins which are superior tadpole attractant chemicals compared to bufotoxins. The resulting aqueous composition is partitioned between water and an organic solvent to isolate the bufagenin compounds from other secretion components such as residual bufotoxins, diacid amides of arginine, catacholamines and indolo-alkyl amines (including but not limited to 5-methoxy 20 N,N-dimethyltryptamine and bufotenine). Suitable solvents include higher alcohols such as n-butanol, chlorinated solvents such as chloroform and dichloromethane, esters such as ethyl acetate, hydrocarbons such as hexane and pentane and ethers such as diethylether, or a mixtures of such solvents. A particularly suitable solvent is ethyl acetate. 25 The solvent fraction is separated from the water fraction and evaporated to dryness to yield a chemical attractant composition. The exact identity of the bufagenin compounds of formula (I) (R 5 = H) and/or the ratio of such compounds may vary depending on the source of the toads, for example, age, species 30 and geographical location, but this does not adversely affect the efficacy of the attractant extract composition.

H:\sxf\Interwoven\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 15 In particular embodiments, the composition is an extract that comprises at least one of marinobufagenin (1), telocinobufagin (2) and bufalin (3), especially marinobufagenin or telocinobufagin. 5 In some embodiments, the bufagenin compounds of formula (I) are esterified with C 7

-C

20 acyl groups including fatty acids or fatty acid derivatives to provide 3-0-esters. In some embodiments, the bufagenin compound is esterified with a C 7 to C 20 or C12 to C 20 acyl group which is either saturated or unsaturated. In some embodiments, the esterified 10 compound of formula (I) is a compound of formula (IA): 0 0 R6 R7 CH3 RR R2 R5- O000 R4 (IA) wherein

R

1 is -OH and R 2 is hydrogen or -OH or R 1 and R 2 taken together form -0-;

R

3 is methyl or -CHO; 15 R 4 is hydrogen or -OH;

R

5 is hydrogen, -C(O)(CH2)tR 9 or -C(O)(CH2)u(CH=CHCH2)v(CH2)wR 9 ;

R

6 and R 7 are both hydrogen or together form =0;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; 20 t is an integer from 10 to 18; u is an integer from 3 to 10; H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 16 v is an integer from 1 to 4; and w is an integer from 3 to 10; wherein u + v + w = 10 to 18. 5 In some embodiments, the bufagenin esters are 3-0-esters of marinobufagenin (4), telocinobufagin (5), bufalin (6), arenobufagin (7), resibufagenin (8) or hellebrigenin (9) in which R 5 is selected from one of -C(O)(CH 2

)

3

CH

3 , -C(O)(CH 2

)

4

CH

3 , -C(O)(CH 2

)

5

CH

3 ,

-C(O)(CH

2

)

6

CH

3 , -C(O)(CH 2

)

7

CH

3 , -C(O)(CH 2

)

8

CH

3 , -C(O)(CH 2

)

9

CH

3 ,

-C(O)(CH

2

)

10

CH

3 , -C(O)(CH 2

)

12

CH

3 , -C(O)(CH 2

)

1 4

CH

3 , -C(O)(CH 2

)

16

CH

3 , 10 -C(O)(CH 2

)

1 8

CH

3 , -C(O)(CH 2

)

7

CH=CH(CH

2

)

5

CH

3 , -C(O)(CH 2

)

7

CH=CH(CH

2

)

7

CH

3 ,

-C(O)(CH

2

)

7

CH=CHCH

2

CH=CH(CH

2

)

4

CH

3 , -C(O)(CH 2

)

7

(CH=CHCH

2

)

3

CH

3 ,

-C(O)(CH

2

)

3

(CH=CHCH

2

)

4

(CH

2

)

3

CH

3 , -C(O)(CH 2

)

3

CH

2 OH, -C(O)(CH 2

)

4

CH

2 OH,

-C(O)(CH

2

)

5

CH

2 OH, -C(O)(CH 2

)

6

CH

2 OH, -C(O)(CH 2

)

7

CH

2 OH, -C(O)(CH 2

)

8

CH

2 OH,

-C(O)(CH

2

)

9

CH

2 OH, -C(O)(CH 2

)

1 0

CH

2 0H, -C(O)(CH 2

)

1 2

CH

2 0H, -C(O)(CH 2

)

1 4

CH

2 0H, 15 -C(O)(CH 2

)

1 6

CH

2 0H, -C(O)(CH 2

)

1 8

CH

2 0H -C(O)(CH 2

)

7

CH=CH(CH

2

)

5

CH

2 0H,

-C(O)(CH

2

)

7

CH=CH(CH

2

)

7

CH

2 0H, -C(O)(CH 2

)

7

CH=CHCH

2

CH=CH(CH

2

)

4

CH

2 0H,

-C(O)(CH

2

)

7

(CH=CHCH

2

)

3

CH

2 0H and -C(O)(CH 2

)

3

(CH=CHCH

2

)

4

(CH

2

)

3

CH

2 0H. The esters may be prepared by well known methods in the art, for example, using acid 20 catalysis, base catalysis or activated carboxylic acids such as activated fatty acids. Suitable acid catalysts include methanolic hydrogen chloride, methanolic sulphuric acid, boron trifluoride methanol and aluminium trichloride. Suitable base catalysts include sodium and potassium methoxide. Suitable activated carboxylic acids such as fatty acids include acid anhydrides, acid chlorides and acid imidazolides such as fatty acid anhydrides, fatty acid 25 chlorides and fatty acid imidazolides. In particular embodiments, the ester is formed using an activated acid anhydride, an acid chloride, or a fatty acid selected from a fatty acid anhydride and a fatty acid chloride. In some embodiments, the attractant composition used in the methods is an extract 30 obtained from toad eggs. In other embodiments, the attractant composition is the parotoid secretion of a toad. In yet other embodiments, the attractant composition is an extract H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 17 obtained from the parotoid secretion of a toad. In yet other embodiments the attractant composition comprises at least one bufagenin compound of formula (I) and/or at least one 3-0-ester that may be prepared by esterification of an extract of the parotoid secretion of a toad with an alkyl carboxylic acid, a primary hydroxyl carboxylic acid, a fatty acid or a 5 primary hydroxyl fatty acid. The toad, toad tadpoles or toad eggs may be any toad, tadpoles or eggs of the family Bufonidae that needs to be controlled. Suitable toads of the family Bufonidae include toads from the genus Rhinella such as Rhinella marina (formerly Bufo marinus), or from 10 the genus Bufo such as Bufo alvarius, Bufo americanus, Bufo arenarum, Bufo asper, Bufo blomberg, Bufo bufo, Bufo bufogargarizans, Bufo formosus, Bufo fowleri, Bufo melanostictus, Bufo peltocephalus, Bufo quercicus, Bufo regularis, Bufo valliceps, Bufo viridis, Bufo vulgaris. In a particular embodiment, the toad of the genus Rhinella to be controlled is a toad of the species Rhinella marina. 15 Also provided is a use of the compositions described above as an attractant for tadpoles of the family Bufonidae, especially tadpoles of the species Rhinella marina. In yet another aspect of the invention there is provided a method of controlling a 20 population of tadpoles of the family Bufonidae in a body of water comprising: a) providing a trapping device to a body of water where the body of water hosts a population of tadpoles from the family Bufonidae; b) baiting the trapping device with a tadpole attractant composition comprising or consisting of one of the following: 25 (i) an extract obtained from the eggs of a toad of the family Bufonidae; (ii) a parotoid secretion of a toad of the family Bufonidae; and (iii) an extract obtained from the parotoid secretion of a toad of the family Bufonidae; and c) harvesting the tadpoles from the trapping device. 30 In another aspect of the invention, there is provided a method of controlling the population H:\sxf\Intrw ovn\NRPortb\DCC\SXF\5228565I.doc-7/06/2013 - 18 of tadpoles of the family Bufonidae in a body of water comprising: a) providing a trapping device to a body of water wherein the water hosts a population of tadpoles of the family Bufonidae; b) baiting the trapping device with a tadpole attractant composition comprising at 5 least one bufagenin compound of formula (I): 0 0 R6 R7 CH3 R8g R3 R2 R5--O R4() wherein

R

1 is -OH and R 2 is hydrogen or -OH or R 1 and R 2 taken together form -0-;

R

3 is methyl or -CHO; 10 R 4 is hydrogen or -OH;

R

5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; 15 t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10; and c) harvesting the tadpoles from the trapping device. 20 The trapping device may be any device in which the tadpoles can enter easily but cannot H:\sxfInterwoven\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 19 exit or can only exit with difficulty. For example, the device may comprise a container of any size or shape that has at least one funnel fitted through a container wall. The wide mouth of the funnel being located on the external surface of the wall and allows the tadpoles to easily find entry into the container, the thin end of the funnel being located 5 internally in the container and having an aperture of a size suitable to allow the tadpoles to pass through it but small enough to make it difficult for the tadpoles to re-enter the funnel once inside the container. Alternatively, the entry device, whether funnel shaped or another shape, may be fitted with a flap that allows entry into the trap but does not allow exit from the trap. The container may also include perforations or apertures in its walls to 10 aid the diffusion of the attractant compound(s) into the water surrounding the container thereby attracting the tadpoles. The perforations or apertures are ideally small enough to prevent the tadpoles leaving the trap. In other embodiments, the container may have larger perforations covered with a material permeable to water and the attractant compounds but not the tadpoles, for example, a mesh material. In yet other embodiments, the trap may be 15 made of a biodegradable material such as a biodegradable plastic material, that will degrade in water over time resulting in dead tadpoles being released back into the water for natural decomposition. In some embodiments, at least a portion of the trap is made of or coated with a dark coloured material, especially a black material. This dark coloured material absorbs heat into the trap resulting in the death of the tadpoles. Biodegradable 20 traps loaded with slow release devise of the invention may be very useful for trapping tadpoles in remote areas where monitoring, maintenance and emptying of traps may not be practical. In such traps, the tadpoles may die in any of the following ways: suffocation, heat, poison and/or starvation. 25 The trap container may vary in size depending on the number of tadpoles likely to be trapped or the intended frequency of harvesting the tadpoles from the trap, and the size of the body of water in which the trap is placed. The number of apertures to allow entry of the tadpoles may also vary depending on features such as the size of the container and the number of tadpoles likely to be trapped. 30 In some embodiments, the composition is an extract of toad eggs, a secretion from the H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -20 parotoid gland of a toad, an extract of the secretion of a parotoid gland of a toad or a composition comprising at least one compound of formula (I) above. In some embodiments, the trap is baited by adding the composition to the water in the trap. 5 In some embodiments, the composition is applied to a device that allows slow release of the compounds of formula (I). For example, the composition may be applied to a water insoluble organic or inorganic matrix, such as a sponge, a piece of glass, a porous glass frit, porous ceramic matrix or a polymeric disc, plate or crumb, for example a rubber disc 10 or crumb, that may be placed in the trapping device. Without wishing to be bound by theory, the compounds of formula (I) are substantially insoluble in water and therefore only slowly disperse into the water in the trap from the surface of the insoluble matrix. 15 In some embodiments, the amount of composition containing the at least one compound of formula (I) used as a bait is between 0.01 mg and 5 g or 6 g, especially 0.1 mg to 1.0 g. In embodiments in which the composition is a secretion from the parotoid gland of the toad, a greater amount of the composition may be required, for example, 0.5 g to 2 g, especially 20 about 1 g. In embodiments in which the composition is an extract of toad eggs or of the parotoid gland secretion, the attractant compounds are concentrated in the composition and a smaller amount is required, for example, 0.1 mg to 0.5 g, especially 0.1 mg to 250 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg or 0.1 mg to 10 mg. 25 In a further aspect of the invention there is provided a sustained release device comprising: a) a water insoluble matrix; b) a composition comprising or consisting of one of the following: i) an extract obtained from the eggs of a toad of the family Bufonidae; ii) a parotoid secretion of a toad of the family Bufonidae; and 30 iii) an extract obtained from the parotoid secretion of a toad of the family Bufonidae.

H:\sxf\Intrw ovn\NRPortb\DCC\SXF\5228565I.doc-7/06/2013 -21 Thus in another aspect of the invention there is provided a sustained release device comprising: a) a water insoluble matrix; and 5 b) a composition comprising at least one bufagenin compound of formula (I): 0 0 R6 R7 CH3 R8g R3 R2 R5--O R4() wherein

R

1 is -OH and R 2 is hydrogen or -OH or R 1 and R 2 taken together form -0-;

R

3 is methyl or -CHO; 10 R 4 is hydrogen or -OH;

R

5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; 15 t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10. 20 The tadpoles may be harvested from the trap and disposed of. The tadpoles in the trap may be dead or alive and this may depend on the time elapsing between setting the trap and H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 22 emptying the trap. In some embodiments, the trap is emptied while the tadpoles are still alive to avoid the release of alarm pheromones from dead or decomposing tadpoles. However, in other embodiments, the trap may be located in a remote area and not emptied regularly. If tadpoles are alive upon harvesting they are killed humanely after removal 5 from the trap. In some embodiments, the sustained release device comprises from about 0.01 mg to 5 g or 6 g of composition comprising the at least one compound of formula (I), especially 0.1 mg to 1 g. In embodiments in which the composition is a secretion from the parotoid gland of 10 the toad, a greater amount of the composition is required, for example, 0.5 g to 2 g, especially about 1 g. In embodiments in which the composition is an extract of toad eggs or of the parotoid gland secretion, the attractant compounds are concentrated in the composition and a smaller amount is required, for example, 0.1 mg to 0.5 g especially 0.1 mg to 250 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg or 0.1 mg to 10 mg. 15 In some embodiments, the trap may be made from or include readily available materials. For example, the trap may be made from a plastic bottle, such as a biodegradable bottle, milk bottle or soda bottle, into which a sustained release device is placed and an entry point is pierced into the bottle or cap of the bottle or a funnel shaped receptacle is fitted to 20 the bottle opening for entry of the tadpoles. In yet another aspect of the invention there is provided a kit comprising: i) a sustained release device of the invention; and ii) an attachment that may be fitted to a container, wherein the attachment is fitted 25 with a funnel shaped cylinder. In this aspect, the sustained release device may be fitted into the container such as a bottle and the attachment fitted to seal the container to form a trap that may be used in the methods of trapping tadpoles described above. 30 Simple bottle traps may be placed in or thrown into water sources which may be infested H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 23 with or are likely to be infested with or are at risk of being infested with cane toad tadpoles. In some embodiments, these simple traps may be dropped from the air into water sources in remote areas. 5 In some embodiments, the composition is an extract from cane toad eggs. In some embodiments, the composition is a parotoid secretion of a toad. In other embodiments, the composition is an extract of the parotoid secretion of a toad. In another aspect of the invention there is provided a composition comprising at least one 10 bufagenin compound of formula (I): 0 0 R6 R7 CH3 R3 H R5-O R4(I) wherein

R

1 is -OH and R 2 is hydrogen or -OH or R 1 and R 2 taken together form -0-;

R

3 is methyl or -CHO; 15 R 4 is hydrogen or -OH;

R

5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH;

R

9 is -CH 3 or -CH 2 OH; 20 t is an integer from 3 to 18; u is an integer from 1 to 10; H:\sxf\Intrw ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 24 v is an integer from 1 to 4; and w is an integer from 1 to 10; wherein the composition is free of hallucinogenic alkaloids. 5 In some embodiments, the composition comprises at least one of marinobufagenin (1), telocinobufagin (2) and bufalin (3). 0 0 0 0 CH, CH, CH3 H C HICH, H CJ O H OH0 HO HO OH OH 0 0 (3) CH3 H H OH HOO C H (3) In other embodiments, the composition comprises at least one 3-0-ester of a bufagenin 10 compound of formula (I): H:\sxf\Intrw ovn\NRPortb\DCC\SXF\5228565I.doc-7/06/2013 - 25 0 0 R6 R7 CH3 R3 H H R1 R R5-O R4(I) wherein

R

1 is -OH and R 2 is hydrogen -OH or R 1 and R 2 taken together form -0-; 5 R 3 is methyl or -CHO;

R

4 is hydrogen or -OH;

R

5 is -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ;

R

6 and R 7 are both hydrogen or together form =O;

R

8 is hydrogen or -OH; 10 R 9 is -CH 3 or -CH 2 OH; t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from 1 to 4; and w is an integer from 1 to 10; 15 especially one of the following compounds: H:\sxf\Intrwovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -26 0 0 0 0 C CH CH, H C H, H O OH0 RO* RO OH OH 0 O O CH, H C3 H OH H OH RO RO H (6) H (7) O O O O CH 0 HC3 C~sHH CH3 : 4H HO H CJ H O H OH RO HRO (8) OH (9)

R

5 is -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 and R 9 is -CH 3 or -CH 2 OH; [3-0-esters of marinobufagenin (4), telocinobufagin (5), bufalin (6), arenobufagin (7), H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 27 resibufagenin (8) and hellebrigenin (9)]. In some embodiments, the at least one compound of formula (I) is derived from an extract. In other embodiments, the compounds of formula (I) may be prepared synthetically from 5 known starting materials or from other bufagenin compounds using known synthetic methods. The compositions are suitable for use in the form of extracts or solutions in solvents such as ethyl acetate, n-hexane, pentane and n-butanol. However, in some embodiments, the 10 compositions may be formulated with other ingredients to provide sustained release over a long period of time, to prevent degradation over time, to control rate of release, and to aid formulation. The compositions may be formulated in dispensers or carriers such as microcapsules, 15 tablets, laminates, capillaries or granules. In particular embodiments, the formulation carrier is a biodegradable carrier such as gypsum, plaster of paris, wax such as bees wax, lanolin, shellac wax, ozocerite, ceresin, montan, polyethylene glycol (carbowax), charcoal, coal or paraffin or a combination thereof. The carrier may enable the composition to be formulated as a solid block or granules from which the attractant is released in a controlled 20 and continuous manner as the carrier degrades or may be released by diffusion out of the carrier. The compositions may also include other additives such as volatility suppressants or enhancers, antioxidants, UV blockers and/or absorbers, antimicrobials, plasticisers, 25 emulsifiers and activated charcoal. Antioxidants such as vitamin E, BHA, BHT, and others can protect the attractants from degradation and may be added in amounts from 0.1% to about 3% by weight. 30 Emulsifiers such as lecithin and modified lecithins, mono and diglycerides, sorbitan monopalmitate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene-sorbitan H:\sxfInterwoven\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -28 monooleate, fatty acids, lipids, etc. provide or improve emulsification properties of the composition and may be added in amounts from 1%-10% by weight. Ultraviolet blockers such as beta carotene or p-aminobenzoic acid protect the components 5 from light degradation and may be added in amounts from 1% to about 3% by weight. Antimicrobials such as potassium sorbate, nitrates, nitrites, and propylene oxide that protect the components from microbial destruction may be added in amounts from 0.1% to about 2% by weight. 10 Plasticizers such as glycerin or soy oil affect physical properties of the composition and may extend its resistence to environmental degradation and may be added in amounts from 0.5% to 4%. 15 Activated charcoal absorbs compounds and releases them in a controlled manner. It can be added in amounts up to 25%, preferably about 5%. Such formulations may be prepared by methods known in the art, for example, melting a wax carrier and adding and mixing with the attractant composition and any other additives 20 and allowing the wax to set. In order that the invention be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples and figures. 25 Brief Description of the Figures Figure 1 is a graphical representation of the cumulative number of tadpoles of cane toads (Rhinella marina) caught in two natural water bodies in tropical northern Australia, using funnel traps baited with the secretion of the parotoid gland of adult cane toads. 30 Figure 2 is a graphical representation showing the trapping rate of a trap using the same H:\sxf\Interwoven\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 29 sponge loaded once with 50 mg of ethyl acetate extract of parotoid secretion of adult cane toad over 4 trials of 24 hours. Figure 3 is a graphical representation showing the Attraction Index of an ethyl acetate 5 extract of cane toad parotoid secretion for cane toad tadpoles (R. marina), and native tree frogs of the species Litoria. nasuta, L. caerulea and L bicolor. Figure 4 is a graphical representation showing the mean percentage of toad tadpoles trapped over a 24 hour period with bufagenins, bufagenin octanoyl esters and bufagenin 10 palmitoyl esters.

H:\sxf\ntr ovn\NRPortb\DCC\SXF\5228565I.doc-7/06/2013 - 30 Examples Example 1: Compositions derived from toad eggs as a tadpole attractant Rhinella marina, formerly Bufo marinus, toad eggs from laboratory-laid clutches were freeze-dried, extracted with methanol, and dried in vacuo. The crude methanol extract was 5 partitioned between n-butanol and water. The n-butanol soluble extract was active in the attractant bioassay, and was defatted with n-hexane. The defatted n-butanol extract was fractionated with semi-preparative HPLC (Agilent Zorbax SB-C8 column, 5 [am, 9.4 x 250 mm, eluting from 90% water/acetonitrile to 100% acetonitrile in 15 min, wash in 100% acetonitrile for 12 min, 3.5 mL/min) to give 26 fractions. 10 These fractions were presented to groups of 20 toad tadpoles (midway through development, at Gosner stages 30-38) in standardised trials to measure attraction and feeding responses (for methods of collection, husbandry and testing are described in Crossland et al., Animal Behaviour, 2011, 82, 775-782). All toad tadpoles came from the toad population on the Adelaide River floodplain, 60 km east of Darwin, in the Northern 15 Territory. At 5-minute intervals after the stimulus was presented (1 mL dropped onto the water surface inside a mesh box in one corner of a 70 x 45 x 9 cm plastic tray, with water 5 cm deep), the number of toad tadpoles within the quarter of the tray closest to the stimulus origin, and the number that were actively feeding (head-down posture, tail wriggling) were counted. Simultaneously-conducted control trials were used to test the statistical 20 significance of any behavioural responses to the stimuli presented. These studies localised behavioural effectors in fractions rich in bufagenins, bufolipins (where the term bufolipin is used to describe natural bufagenin compounds esterified at the 3-hydroxy group with a fatty acid) and fatty acids. Statistical tests show that in all cases with >50% of tadpoles exhibiting either attraction or feeding were significantly [P < 0.05] 25 different from control trials, even after Bonferroni correction. Example 2: Compositions derived from toad parotoid secretions as a tadpole attractant Funnel-traps made from rectangular plastic boxes with holes cut on two diagonally 30 opposite sides to accommodate plastic funnels (holes 6.5 cm diameter; funnel length 5 cm, minimum internal funnel diameter 13 mm) were used. To obtain toxin for laboratory and H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 -31 field trials, an adult toad was held in gloved hands beneath a protective sheet of glass, and the shoulder (parotoid) glands gently squeezed to expel the toxin onto the underside of the glass. For field trials the exudate was wiped off onto a glass microscope slide and weighed (2 ± 0.1 g per slide, requiring about 4 toads) and one slide per funnel-trap was used as bait, 5 replaced daily. Six traps were placed at equal intervals around the margins of each of two natural waterbodies on the Adelaide River floodplain (Pond 1 = 84 m perimeter, 12'34'39"S, 131'19'4"E; Pond 2 = 78 m perimeter, 12'45'6"S, 131'29'8"E). Traps were placed in shallow water (13 cm), with shadecloth covers to prevent overheating at midday. On the 10 morning of day 1 the parotoid secretion attractant was added. Traps were then checked and cleared every 24 h for the next 7 days; all trapped individuals were removed and held in captivity to prevent them being re-captured. If toad tadpoles were too numerous to count by hand, 100 representative specimens were weighed and estimated capture rates based on total mass divided by mean mass per toad tadpole. Daily (diurnal and nocturnal) visual 15 surveys for toad tadpoles, metamorph cane toads, and fishes before, during and after the trapping sessions were conducted. Funnel traps baited with toad toxin (exudate from the parotoid glands of adult cane toads) rapidly caught tens of thousands of toad tadpoles (Figure 1), and very little else. The ponds contained thousands of native fish (>5,000 per pond, based on visual surveys before and 20 after our trapping sessions) but the toxin-baited traps caught <30 fish (all trout gudgeons, Mogurnda mogurnda). Thus, vertebrate bycatch constituted <0.1% of animals caught (42,000 toad tadpoles, 27 fish). The traps also captured 24 invertebrates, mostly water scorpions and beetles (both present in each pond in thousands). The rapid reduction (to zero) in capture rates suggests that most toad tadpoles in the ponds were caught in the 25 traps. Because metamorph toads are diurnal, and are restricted to pond margins from the time of emergence until rain falls, the numbers of recruiting metamorphs can be accurately determined by visual counts. Our surveys detected no metamorph toads emerging from the ponds in the two weeks following trapping sessions.

H:\sxf\ntr ovn\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 32 Example 3: Selectivity of attractant for cane toad tadpoles The ponds trapped in Example 2 did not contain tadpoles of native frogs, therefore additional laboratory trials to clarify the species-specificity of that attractant response were conducted. A funnel trap containing 1 g of parotoid exudate (obtained from 2 to 4 toads, as 5 described in Example 2) on a glass slide was placed in a large circular wading pool (2.4 m diameter, 15 cm deep, 700 litres) containing 100 tadpoles either of cane toads, or of native species (either Litoria caerulea, L. rubella, or L. rothii, all obtained by collecting egg masses in local waterbodies, and raising tadpoles in captivity until testing). An adjacent pool containing an identical but unbaited funnel-trap (i.e., containing a glass slide without 10 toxin) served as a control. Two replicates were run for treatment and control for each of the four species (i.e., cane toads, and the three treefrogs). In each case, the numbers of tadpoles trapped within 24 h after the trial commenced were counted. These trials showed that the parotoid secretions of adult cane toads attracted toad tadpoles into traps, but repelled the tadpoles of native frog species. The numbers of toad tadpoles 15 captured in toxin-baited traps was higher than in control traps (totals 186 vs 41; against a null of equal numbers, X2 = 92.62, 1 df, P < 0.001) whereas the reverse was true for 2 2 tadpoles of Litoria caerulea (39 vs 57; X2 = 3.38, 1 df, P = 0.06), L. rothii (53 vs 117; x 24.09, 1 df, P < 0.001), and L. rubella (39 vs 106; X2= 30.96, 1 df, P < 0.001). 20 Example 4: Duration of Attractant Effect Laboratory studies in which the chemical attractant was provided on day 1 and at the end of each day for three days, the number of tadpoles trapped were counted. Capture rates per 100 tadpoles per day in large wading pools fell from 93% for fresh parotoid secretion, to 69% for one-day-old secretion (kept in water throughout the intervening period), to 54% 25 for two-day-old secretion, to 45% for three-day-old secretion. This study shows that the parotoid secretion attractant can remain active for at least three days. Example 5: Attractant activity of compounds found in Cane Toad parotoid secretion Using a laboratory study in test tanks, the attractant activity of compounds present in toad 30 eggs and toad parotoid secretion was assessed. A tank containing 12 L of water was divided into 4 quarters and 20 toad tadpoles were added to the tank. Samples (0.1 mg) of H:\sxf\Interwoven\NRPortbI\DCC\SXF\5228565I.doc-7/06/2013 - 33 test compound was placed on a filter disc and placed in one quarter of the tank. After 5 minutes, the number of tadpoles present in the quarter containing the compound disc and the number exhibiting feeding behaviour in that quarter were counted. The results are shown in Table 1. 5 Table 1 compound # tadpoles # tadpoles with % tadpoles % tadpoles with in quarter feeding behav. in quarter feeding behav. Control 1 6 0 30 0 Control 2 5 0 25 0 parotoid butanol extract 15 12 75 60 parotoid water extract 13 9 65 45 marinobufotoxin 5 0 25 0 marinobufagenin 17 8 85 40 telocinobufagin 11 4 55 20 suberoyl arginine 6 0 30 0 pimeloyl arginine 5 0 25 0 Example 6: Concentration of Attractant An ethyl acetate extract derived from the parotoid secretion was dissolved in MeOH and 10 was loaded onto small pieces of kitchen sponge (Woolworth homebrands) with concentration range of 0.01 - 10 mg. These were tested in the small tray divided into quarters and using 20 tadpoles as outlined in Example 5. The result showed that tadpoles can detect the bufagenin loaded onto pieces of sponge, with the number of tadpoles present in the stimulus quarter increasing with the increase in the extract concentration (Table 2), 15 although the excited feeding response stayed more or less the same with different concentrations.

H:\sxf\Intrwovn\NRPortb\DCC\SXF\5228565I.doc-7/06/2013 - 34 Table 2 Treatment Mean % SE Mean % SE tadpole in tadpole with quarter feeding behav Control (no sponge) 35 3 0 0 Control (small sponge) 35 3 0 0 Control (med sponge) 37 5 0 0 Control (large sponge) 34 3 0 0 0.01 mg extract 47 6 4 4 0.05 mg extract 44 6 25 2 0.1 mg extract 55 5 25 2 0.5 mg extract 62 8 22 6 1.0 mg extract (batch 1) 69 4 47 8 1.0 mg extract (batch 2) 55 3 29 5 5.0 mg extract 60 2 27 5 10 mg extract 57 9 22 5 Example 7: Trapping Trials with Ethyl Acetate Extract Using a wading pool and a funnel trap, the number of cane toad tadpoles trapped over 24 5 hours was assessed using sponges loaded with 50 mg, 100 mg and 200 mg parotoid secretion ethyl acetate extract. The trial using the 50 mg sponge was repeated. The results are shown in Table 3.

H:\sxfInterwoven\NRPortbl\DCC\SXF\5228565I.doc-7/06/2013 - 35 Table 3 Treatment % tadpoles % tadpoles trapped trapped at 24 hr at 24 hours in in control trap baited trap 50 mg extract 19.5 ±6.5 85.5 ±3.5 100 mg extract 40 95 200 mg extract 41 90 Example 8: Longevity of Attraction of Extract The trap test of Example 7 was repeated 4 times using the same sponge to assess the 5 longevity of the attractant extract. The results are shown in Figure 2. This figure shows after four uses, there was no decline in the trapping of the tadpoles and therefore no decline in attractant efficacy. Example 9: Specificity of Attraction of Extract 10 The wading pool experiments described in Example 3 were repeated with sponge baited with 50 mg ethyl acetate extract (bufagenin). The number of tadpoles of each species trapped were counted at 6 hours and 24 hours. The % trapped in the baited trap minus the % trapped in the control trap was used as an Attraction Index, a positive score is indicative of attraction. The results are shown in Figure 3. 15 After 6 hours there is evidence of attraction for cane toad tadpoles in the baited traps, but very few native tadpoles have entered the traps. After 24 hours, many more toad tadpoles are trapped in the baited trap than in the control trap. The reverse pattern occurs for Litoria caerulea and L. nasuta, while for L. bicolor, there were low numbers in the baited 20 trap. The small number of L. bicolor trapped would not translate to a significant effect on the native population. Example 10: Preparation of bufagenin-3-0-esters The parotoid secretion from 60 toads was collected directly in to water (300 mL) and after 25 30 mins was extracted with ethyl acetate (3 x 250 mL). The resulting ethyl acetate extract H:\sxf\ntr ovn\NRPortb\DCC\SXF\5228565I.doc-7/06/2013 - 36 was concentrated in vacuo to yield an ethyl acetate extract (680 mg). A sample of this extract (1 mg) dissolved in methanol (1 mL) was analyzed by high performance liquid chromatography equipped with a diode array detector and a mass spectrometer detector (HPLC-DAD-MS) to reveal a mixture of bufagenins: -53% marinobufagenin, -24% 5 bufalin, ~18% telocinobufagin, -2% arenobufagin and -2% resibufagenin. Samples of the ethyl acetate extract (50 mg in 1 mL dry dichloromethane) were treated with solutions consisting of either i) octanoic acid (54 mg in 1 mL dry dichloromethane, 3 mole equivalent) plus N,N'-dicyclohexylcarbodiimide (77 mg in 1 mL dry dichloromethane, 3 mole equivalent) plus 4-dimethylaminopyridine (46 mg in 1 mL dry 10 dichloromethane, 3 mole equivalent). or ii) palmitic acid (96 mg in 1 mL dry dichloromethane, 3 mole equivalent), plus N,N'-dicyclohexylcarbodiimide (77 mg in 1 mL dry dichloromethane, 3 mole equivalent) plus 4-dimethylaminopyridine (46 mg in 1 mL dry dichloromethane, 3 mole equivalent). The solutions were then stirred at room temperature overnight (12 h), at which time the solutions were eluted through a filter paper 15 (to remove residual dicyclohexylurea). The crude esterified product was purified by silica flash chromatography eluting with a 20% stepwise gradient from hexane to ethyl acetate, to yield mixtures of bufagenin octanoyl esters and bufagenin palmitoyl esters.The esterified bufagenins ere not separated into individual compounds but were used in field trials as a mixture of either octanoyl or palmitoyl esters. 20 These mixtures of bufagenin esters were analysed by HPLC-DAD-MS and determined to be mixtures of marinobufagenin-3-0-ester, bufalin-3-0-ester, telocinobufagin-3 -0-ester, arenobufagin-3-0-ester and resibufagenin-3-0-ester with a ratio comparable to that of the unesterified ethyl acetate starting material. The mixtures of bufagenin octanoyl esters and bufagenin palmitoyl esters were used as is in field trials. 25 Example 11: Release rates of bufagenins and bufagenin esters from a ceramic mixtures Samples of the parotoid secretion ethyl acetate extract (2 mg in 0.4 mL methanol ), and the corresponding octanoyl esters (bufagenin-3-0-octanoyl esters) (2.5 mg in 0.4 mL 30 methanol), and the corresponding palmitoyl esters (bufagenin-3-0-palmitoyl esters) (3 mg in 0.4 mL methanol) were applied by syringe to individual porous ceramic matrices H:\sxf\Intr ovn\NRPortb\DCC\SXF\5228565I.doc-7/06/2013 - 37 (purchased as -4-5 g ceramic filter stones from aquarium suppliers). The porous ceramic matrix samples were then air dried before use. The release rate of the parotoid secretion ethyl acetate extract, the corresponding octanoyl esters (bufagenin-3-0-octanoyl esters), and the corresponding palmitoyl esters (bufagenin 5 3-0-palmitoyl esters) from the porous ceramic matrix was analysed by immersing an impregnated matrix into a container of water (20 mL) with 25 [iL water sampling at 2, 12, 24, 48, 72 and 96 h. Aliquots (1 [tL) of each water sample were analysed by ultra high performance liquid chromatography with a diode array detector (UPLC-DAD, monitoring at 298 nm) (Zorbax RRHD SB-C 8 Agilent 1.8 im 2.1 x 50 mm column, 0.417 mL/min 10 gradient elution from 90% water/acetonitrile to 100% acetonitrile in 2.5 min, followed by a 1.5 min hold at 100% acetonitrile, with a constant 0.01% trifluoroacetic acid as modifier). This analytical method is capable of detecting bufagenins and bufagenin esters at concentrations of 1 [tg/mL in the original water samples. With 24 h -75% of bufagenins were released into the surrounding water, leading to -85% 15 at 96 h. By comparison, even after 96 h bufagenin-3-0-octanoyl esters and bufagenin-3 O-palmitoyl esters release rates were below the detection limit of the analytical method (<1 [tg/mL). The release of bufagenin-3-0-octanoyl esters and bufagenin-3-0-palmitoyl esters remained below the detection limit even after 3 weeks immersion in water. 20 Example 12: Bufagenins and bufagenin esters as tadpole attractants The trapping of cane toad tadpoles (Rhinella marina) bred in captivity (100 tadpoles per pool), was assessed over a 24 hour period using childrens wading pools (2.5 m diameter) and funnel traps baited with a porous ceramic matrix impregnated with one of parotoid secretion ethyl acetate extract (bufagenins) (2mg), or the corresponding octanoyl esters 25 (bufagenin-3-0-octanoyl esters) (2.5 mg), or the corresponding palmitoyl esters (bufagenin-3-0-palmitoyl esters) (3 mg). The percentage of tadpoles trapped in each pool is shown in Table 4 and Figure 4: H:\sxf\Intrwovn\NRPortb\DCC\SXF\5228565I.doc-7/06/2013 - 38 Table 4: Mixture Mean % cane toad tadpoles trapped at time points (hours) Oh 1h 2h 3h 4h 5h 6h 24h Bufagenins 0 32 59 81 91 93 95 100 Octanoyl 0 8 54 64 77 82 85 100 esters Palmitoyl 0 2 11 22 34 56 69 96 esters Although the release rate of the bufagenin esters is very low compared to that of the 5 unesterified bufagenins, the bufagenin esters showed the same efficacy in trapping tadpoles after 24 h. Significantly, the low release rate ensures that bufagenin ester baits last longer, and may be of repeated use.

Claims (16)

1. 3. The method according to claim I or claim 2 wherein the composition is derived from toad eggs of the family Bufonidae. 15
2. 4. The method according to claim I or claim 2 wherein the composition is derived from a secretion from the parotoid gland of a toad of the family Bufonidae.
3. 5. The method according to claim 1 or claim 2 wherein the composition consists of 20 secretion from the parotoid gland of a toad of the family Bufonidae.
4. 6. The method according to claim 4 wherein the composition is an extract derived from a secretion from the parotoid gland of a toad of the family Bufrnidae. 25 7. The method according to any one of claims I to 6 wherein the toad of the family Bufonidae is a toad of the species Rhinella marina.
5. 8. A method of controlling a population of tadpoles of the family Bufonidae in a body of water comprising: 30 a) providing a trapping device to a body of water where the body of water hosts a population of tadpoles from the family Bufonidae; H:\s\Inerwven\R~oiblDCC\XF\22865_.doe:-7/06/20I3 -41 b) baiting the trapping device with a tadpole attractant composition comprising or consisting of one of the following: (i) an extract obtained from the eggs of a toad of the family Bujonidae; (ii) a parotoid secretion of a toad of the family Bufonidae; and 5 (iii) an extract obtained from the parotoid secretion of a toad of the family Bufonidae; and c) harvesting the tadpoles from the trapping device.
6. 9. A method of controlling the population of tadpoles of the family Bufonidae in a 10 body of water comprising: a) providing a trapping device to a body of water wherein the body of water hosts a population of tadpoles of the family Buftnidae; b) baiting the trapping device with a tadpole attractant composition comprising at least one bufagenin compound of formula (1): 0 0 R6 R R3 HRR R2 RS9 15 R(1) wherein R 1 is -01-O and R 2 is hydrogen or -01-1 or R 1 and R2 taken together form -O-; R 3 is methyl or --- CHO; R4 is hydrogen or -01-1 20 R5 is hydrogen, --- C(O)(CH 2 )tR 9 or -C(O)(C[H2)u(CH=CHCH 2 )v(CH 2 )wR 9 ; R 6 and R 7 are both hydrogen or together forn =0; -42 R 8 is hydrogen or -O-I R 9 is -CH3 or -CH 2 OH; t is an integer from 3 to 18; u is an integer from I to 10; 5 v is an integer from I to 4; and w is an integer from I to 10; and (c) harvesting the tadpoles from the trapping device.
7. 10. The method according to claim 8 or claim 9 wherein the tadpole from the family 10 Bufonidae is a cane toad tadpole of the species Rhinella marina.
8. 11. The method according to any one of claims 8 to 10 wherein the composition is a sustained release composition or is applied to a device that provides sustained release. 15 12. Use of a composition comprising or consisting of one of the following: a) an extract obtained from the eggs of a toad of the family Bufrnidae; b) a parotoid secretion of a toad of the family Bufbnidae; and c) an extract obtained from the parotoid secretion of a toad of the family Bifonidae; 20 as an attractant for tadpoles of the family Bufonidae.
9. 13. Use of a composition comprising at least one bufagenin compound of formula (I): H:\s\Inerwven\R~oiblDCC\XF\22865_.doe:-7/06/20I3 43 R6 R7 H3 H R R2 R5-0 R4,(I wherein R-1 is -OH- and R2 is hlydrogen or -OH- or Ra and R2 t aken together form -O-; R3 is methy I or -CH0; 5 R4, is hydrogen or -OH;1 R 5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(C H 2 )u(C H=C H [H 2 )(CH 2 )wR 9 ; R 6 and R7 are both hydrogen or together form =0: Rs is hydrogen or -01; R 9 is -CH 3 or -CH 2 OH; 10 t is an integer from 3 to 18; u is an integer from 1 to 10; v is an integer from I to 4; and w is an integer from I to 10; as an attractant for tadpoles of the family Buqfonidae. 15
10. 14. Use according to claim 12 or claim 13, wherein the tadpole of the family Bufonidae is from the species Rhinella marina.
11. 15. A sustained release device comprising: 20 a) a water insoluble matrix; b) a composition comprising or consisting of one of the following: H:\s\Inerwven\R~oiblDCC\XF\22865_.doe:-7/06/20I3 -44 i) an extract obtained from the eggs of a toad of the family Bifonidae; ii) a parotoid secretion of a toad of the family Bufonidae; and iii) an extract obtained from the parotoid secretion of a toad of the family Bufonidae.
12. 16. A sustained release device comprising: a) a water insoluble matrix; and b) a composition comprising at least one bufagenin compound of formula (I): R6 R, CH3) R R, R2 R50 R4() 10 wherein R, is -OH and R 2 is hydrogen -OH or R, and R2 taken together form --0-; R3 is methyl or -CH0; R 4 is hydrogen or -OH; R 5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(C H 2 )u(CH=C CH 2 )v(CH 2 )wR 9 ; 15 R 6 and R 7 are both hydrogen or together form =0: Rs is hydrogen or -OH; R 9 is -CH 3 or -CH2OH; t is an integer from 3 to 18; u is an integer from I to 10; 20 v is an integer from I to 4; and w is an integer from I to 10. H:\s\Inerwven\R~oiblDCC\XF\22865_.doe:-7/06/20I3 -45
13. 17. A sustained release device according to claim 15 or claim 16 wherein the water insoluble matrix is a sponge. 5 18. A sustained release device according to claim 15 or claim 16 wherein the water insoluble matrix is a porous glass frit or a ceramic matrix.
14. 19. A composition comprising at least one bufagenin compound of formula (1): 0 0 RS. Ry, H3 R3 H R4 (I) 10 wherein R 1 is -OH and R2 is hydrogen or -OH or R 1 and R 2 taken together form -0-; R3 is methyl or -CHO; R 4 is hydrogen or -OH; R 5 is hydrogen, -C(O)(CH 2 )tR 9 or -C(O)(CH 2 )u(CH=CHCH 2 )v(CH 2 )wR 9 ; 15 R 6 and R- are both hydrogen or together form =0; Rs is hydrogen or -O-I R 9 is -CH 3 or -CH 2 OH; t is an integer from 3 to 18; u is an integer from I to 10; 20 v is an integer from I to 4; and w is an integer from I to 10; and wherein the composition is free of hallucinogenic alkaloids. H:\d\Iterovn\N~orblDCC\SXF\5228565_ .doe:-7/06/20I3 -46 20. The composition according to claim 19 which is an extract derived from a parotoid secretion of a toad of the family Bufonidae. 5 21. The composition according to claim 19 or claim 20 comprising at least one of the following compounds: o 0 o 0 O CH, H CHI H OH HO H .OH OH (2) (1) 0 CHs H OHi HO; H (3)
15. 22. A composition comprising at least one 3-O-ester of a bufagenin compound of 10 formula (1): H:\s\Inerwven\R~oiblDCC\XF\22865_.doe:-7/06/20I3 47 R6 R7 CH3 R3 1 R, R2 R5-0 R4() wherein R 1 is -OH and R2 is hydrogen or -OH or R1 and R2 taken together form -0-; 5 R 3 is methyl or -CHO; R4 is hydrogen or -oH[1; R~s is --- C(O)(CH2)tR 9 or -C(O)(CH2)u(CH=CHCH2)v(CH2)wR9; R 6 and R 7 are both hydrogen or together form =0; Rs is hydrogen or --- OH; 10 R 9 is -C-1 3 or -CH2O1H; t is an integer from 3 to 18; u is an integer from I to 10; v is an integer from I to 4; and w is an integer from I to 10. 15
16. 23. A composition according to claim 22 comprising at least one of the following compounds: H:\s\Inerwven\R~oiblDCC\XF\22865_.doe:-7/06/20I3 -48 0 0 0 0 CH CH, CH, H CH, H H, O H OH RO Rs OH OH '4) (5) 0 " 0 CH CH, CH, H H H. OH 0 H ()H C7) /* 0 0 0 0 CH, Cs CH HCHOH O. OH H R,.O i (s)OH (9) wherein RZ5 is -- C(O)(CH2,),R9 or -C('(O)(CH2)u,(C'H=CHCH2)v,(CH2)wR9 and R9 is -- CH3 or -CH2OH4. 5 24. A composition according to any one of claims 19 to 23 formulated for sustained release. -49 25. A kit comprising: i) a sustained release device according to any one of claims 15 to 18; and ii) an attachment that may be fitted to a container, wherein the attachment is fitted 5 with a funnel shaped cylinder.