NZ577913A - Microencapsulated nitrite salts for poison bait in vertebrate pest control - Google Patents

Abstract

Disclosed is a composition comprising microencapsulated alkali metal salts of nitrite for vertebrate pest control. Also disclosed is a non-toxic paste composition for vertebrate pest control product comprising 10 percent (w/w) microencapsulated sodium nitrite.

Description

Patents Form No. 5 *10059382483* IPPC ref. C01.008NZ Patents Act 1953 Complete Specification We, Charles Thomas EASON of 4 Arkley Avenue, Pakuranga, Auckland, New Zealand, and Duncan Brown MACMORRAN of 79 Jamieson Road, RD2 Pukekohe, New Zealand, hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in the following statement: 1 INTELLECTUAL PROPERTY OFFICE OF N.Z. 21 SEP 2010 RECEIVED! C01.008NZ III Received at IPONZ on 12 January 2011 FORMULATIONS FOR VERTEBRATE PEST CONTROL FIELD OF INVENTION The invention relates to improved formulations of alkali metal salts of nitrite for use as vertebrate pest control agents. In 5 particular, the invention relates to improved formulations of sodium nitrite for use in the control of feral pigs.

BACKGROUND ART Feral pigs (Sus scrofa) are found in abundance in certain areas of New Zealand and can be an agricultural pest, responsible for 10 damage to crops and pasture, and the predation of lambs. Numbers can greatly fluctuate and when numerous, because of their wide-ranging habits, they frequently come into close contact with domestic stock.

Because of their susceptibility to tuberculosis and foot and 15 mouth disease, feral pigs retain a high potential to act as vectors of these diseases. Feral pigs also cause considerable damage to New Zealand's native species and are a likely vector for kauri root collar rot.

Hunting, shooting, fencing and trapping are inadequate methods 20 for the control of populations of feral pigs in remote areas, or areas which are difficult to access. However, feral pigs are also difficult to poison, partly because of their large size.

As a general rule larger doses of a toxic agent are needed for the control of feral pigs than are needed for the control of 25 smaller vertebrate pests. Poisoned baits are therefore employed to encourage the ingestion of larger doses by the target animal.

The use of poisoned baits has become a widely accepted method for the control of feral pigs in rural communities (O'Brien et al, 1986; Choquenot et al, 1996). For example, gelatin capsules 30 containing powdered sodium monofluororacetate (1080) may be inserted into suitable bait, e.g. apples, potatoes or meat (O'Brien et al 1986), and left for the target animal to consume. 2 Received at IPONZ on 12 January 2011 C01.008NZ III The use of 1080 for the control of feral pigs has a number of limitations. The handling of 1080 presents risks to users (Eason, 1989). Despite the use of baits, doses consumed by the target animal may be inadequate. Furthermore, the dose-response 5 relationship for 1080 in feral pigs is inconsistent.

The use of known alternatives to 1080, such as warfarin and yellow phosphorus (sold as CSSP), either do not overcome these limitations, or raise additional concerns related to animal welfare (Sharpe and Saunders, 2004; Cowled and O'Connor, 2004) 10 or residues (Eason and Henderson, 1991).

Winks et al (1950) have reported the toxicity to pigs of sodium nitrite following the heavy mortality of pigs fed on soup prepared by cooking beef and offal in well water.

Hoorens and Thoonen (1961) have also reported the poisoning of 15 pigs attributed to nitrite intoxication caused by drinking water from wells containing blue-green algae and from silos with a high amount of nitrite.

More recently Staples et al (2008) have disclosed nitrite salts as toxic agents for use in the control of feral pigs. In an 20 example the toxic agent is presented in the form of poisoned bait comprising a shaped, solid carrier material with the toxic agent distributed throughout the carrier material.

It is an object of the present invention to provide an improved presentation of nitrite salts particularly suited for use in the 25 control of vertebrate pests and the preparation of poisoned baits or to at least provide a useful choice.

STATEMENT OF INVENTION In a first aspect the invention provides a vertebrate pest control product comprising a microencapsulated alkali metal salt 30 of nitrite.

Preferably, the alkali metal salt of nitrite is sodium nitrite. 3 C01.008NZ III Received at IPONZ on 12 January 2011 Preferably, the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.025 to 0.5. More preferably, the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.05 to 0.25. Most 5 preferably, the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.05 to 0.07.

Preferably, the encapsulating material is selected from the group consisting of: enteric coating material and lipophilic coating material. More preferably, the encapsulating material 10 is a water insoluble, digestible substance. Most preferably, the water insoluble, digestible substance is zein.

Preferably, the encapsulating material includes one or more organoleptic compounds. More preferably, the encapsulating material includes one or more organoleptic compounds selected to 15 promote the palatability to the vertebrate pest of the microencapsulated alkali metal salt of nitrite.

Preferably, the vertebrate pest is selected from the group consisting of: feral pigs, possums and rats.

In a first embodiment of the first aspect the invention provides 20 a vertebrate pest control product comprising microencapsulated sodium nitrite (MSN) where the encapsulating material is zein at 5% (w/w) of the microencapsulated sodium nitrite.

In a second embodiment of the first aspect of the invention, the vertebrate pest control product comprises 10 % (w/w) MSN 25 dispersed in a non-toxic paste composition.

In a second aspect the invention provides the use of an effective amount of a microencapsulated alkali metal salt of nitrite in the preparation of a poisoned bait for the control of a vertebrate pest.

Preferably, the alkali metal salt of nitrite is sodium nitrite.

Preferably, the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.025 to 0.5. More C01.008NZ III Received at IPONZ on 12 January 2011 preferably, the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.05 to 0.25. Most preferably, the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.05 to 0.07.

Preferably, the encapsulating material is selected from the group consisting of: enteric coating material and lipophilic coating material. More preferably, the encapsulating material is a water insoluble, digestible substance. Most preferably, the water insoluble, digestible substance is zein.

Preferably, the poisoned bait includes one or more organoleptic compounds. More preferably, the poisoned bait includes one or more organoleptic compounds selected to promote the palatability to the vertebrate pest of the poisoned bait.

Preferably, the vertebrate pest is selected from the group 15 consisting of: feral pigs, possums and rats.

Preferably, the amount of microencapsulated alkali metal salt of nitrite used in the preparation of the poisoned bait provides a concentration in the poisoned bait in the range 2.5 to 20% (w/w) of the microencapsulated alkali metal salt of nitrite. More 20 preferably, the amount of microencapsulated alkali metal salt of nitrite used in the preparation of the poisoned bait provides a concentration in the poisoned bait in the range 5 to 15% (w/w). Most preferably, the amount of microencapsulated alkali metal salt of nitrite used in the preparation of the poisoned bait 25 provides a concentration in the poisoned bait in the range 7.5 to 12.5% (w/w).

In a third aspect the invention provides a poisoned bait for the control of a vertebrate pest prepared by the use of the third aspect of the invention.

In a fourth aspect the invention provides a microencapsulated alkali metal salt of nitrite manufactured for use in the preparation of the vertebrate pest control product of the first aspect of the invention.

C01.008NZ III Received at IPONZ on 12 January 2011 In the description and claims of this specification the following acronyms, terms and phrases are intended to have the meaning indicated: "213 paste" means a non-toxic composition consisting of peanut 5 butter, mixed grains, ground maize and a dye.

"BAIT-RITE™" means a composition consisting of MSN distributed throughout a non-toxic paste prepared to provide a concentration of 10% (w/w) of sodium nitrite (NaN02) in the composition.

"Digestible" means, with reference to the encapsulating 10 material, becoming friable or soluble in the conditions of the stomach.

"Microencapsulated" means, with reference to an alkali metal salt of nitrite, that the formulation of the alkali metal salt of nitrite is in a granular or powdered form and comprises 15 particles of the alkali metal salt of nitrite coated with an encapsulating material.

"MSN" means microencapsulated sodium nitrite (NaN02) "Pellets" means, with reference to the form of the vertebrate pest control product, solid bodies prepared by compacting a dry 20 mix of the microencapsulated alkali metal salt of nitrite with or without the addition of binders. In this context the term "pellets" is used synonymously with the terms "slugs", "tablets", etc.

"Rodenticide" means a vertebrate pest control product for use in 25 rodent control.

"Vertebrate pest control product" means a formulation of a toxic agent packaged and identified for use in vertebrate pest control.

"Water insoluble" means, with reference to a substance, that the 30 substance is substantially insoluble in water at neutral pH at 25 °C, remaining impervious to water when applied as a coating. 6 C01.008NZ III Received at IPONZ on 12 January 2011 The term "feral" is used synonymously with the term "wild".

The term "pig" is used synonymously with the terms "boar", "hog", "swine", "sow", etc.

The terms "first", "second", "third", etc. used with reference 5 to elements, features or integers of the subject matter defined in the Statement of Invention and Claims, or when used with reference to alternative embodiments of the invention are not intended to imply an order of preference.

Where values are expressed to one or more decimal places 10 standard rounding applies. For example, 1.7 encompasses the range 1.650 recurring to 7.499 recurring.

Exemplary embodiments of the invention will now be described in detail with reference to the Figures of the accompanying drawings pages.

BRIEF DESCRIPTION OF FIGURES Figure 1. Mean time to death for pigs fed bait containing different presentations of microencapsulated sodium nitrite (Study 2, Table 4).

Figure 2. Possum abundance at treatment site (pre-toxin, left column of each pair; post-toxin, right column of each pair).

Figure 3. Bait boxes used in trial.

DETAILED DESCRIPTION Sodium nitrite (NaS03; SN) is a meat preservative, which is found in the normal human diet at very low concentrations. As a 25 food additive it serves a dual purpose since it both alters the colour of preserved fish and meats and also prevents growth of Clostridium botulinum, the bacterium which causes botulism.

Sodium nitrite (E250) may only be used as a mixture with table salt (sodium chloride; NaCl) containing at most 0.6% (w/w) sodium nitrite. Potassium nitrite (E249) is used in the same way. Both nitrites can be added to meat products including bacon, ham, saveloys (but not sausage or sausage meat), luncheon, salami, corned silverside and hamburger. 7 Received at IPONZ on 12 January 2011 C01.008NZ III The concentration of SN being researched in baits to kill possums is of a much higher concentration that that found in any product consumed by humans. Its use as a poison for killing possums is a very recent phenomenon where it is observed to 5 induce methaemoglobinaemia, MtHb (Eason et al 2010a and 2010b).

Death following ingestion of MSN is humane, somewhat slower, but more akin to that seen with cyanide or PAPP. From a welfare perspective it is preferable to other toxins with questionable humaneness used for killing mammalian pests (Littin et al (2000 10 and 2002); Sherley (2004 and 2007)).

A formulation of microencapsulated sodium nitrite has been developed. This formulation is distinguished from the formulation described by Staples et al (2008) where the sodium nitrite is distributed throughout a carrier material.

Without wishing to be bound by theory it is believed the formulation of sodium nitrite in a microencapsulated form promotes ingestion of the toxic agent (sodium nitrite) at a sufficient rate and in a sufficient dose to promote a rapid increase in methemoglobin levels.

A rapid increase in methemoglobin levels is required for death of the target animal to occur. It is contemplated that the rate of ingestion and subsequent absorption may be further enhanced by formulation of the microencapsulated sodium nitrite in a suitably flavoured carrier material and co-administration with 25 an agent (proton pump inhibitor) to reduce the acidity of the stomach.

The microencapsulation of food ingredients is a well established technology (Vistrup, 2001). Known methods are readily adaptable to the preparation of microencapsulated sodium nitrite.

In a preferred embodiment the invention resides in a formulation of sodium nitrite that mitigates the adverse palatability of the toxic agent and consequential feeding deterrence and promotes rapid and sufficient ingestion of the toxic agent by the 8 C01.008NZ III Received at IPONZ on 12 January 2011 vertebrate pest to ensure a rapid and lethal increase in methemoglobin levels.

The formulation of microencapsulated sodium nitrite may be prepared by a number of methods, including fluid bed drying. A 5 preferred encapsulant for use in the preparation of the formulation is the major storage protein of corn, zein.

In its pure form the encapsulant is odourless, tasteless, hard, water insoluble and edible with accepted use in processed food and pharmaceuticals being classified as Generally Recognised As 10 Safe (GRAS) by the US Food and Drug Administration (USFDA).

Selection of zein as the encapsulant is particularly advantageous in the preparation of microencapsulated sodium nitrite and the use of the formulations for vertebrate pest control. Due to the small particle size of the sodium nitrite 15 used in the preparation it can be difficult to achieve complete coverage of each particle as required to mitigate the adverse palatability of the sodium nitrite and consequential feeding deterrence that might otherwise arise.

Once ingested the encapsulant is required to be capable of being 20 rapidly broken down in the stomach of the target vertebrate pest. However, the encapsulant must also be water insoluble to enable its incorporation into a range of baits routinely employed in vertebrate pest management.

The use of zein as an encapsulant was found to be particularly 25 suitable for use as an encapsulant in the context of preparing formulations for invertebrate pest control.

PREPARATION OF MICROENCAPSULATED SODIUM NITRITE (MSN) Method 1 Ten parts by weight of a coating mixture consisting of 10% (w/w) 30 zein, 10% (w/w) water and 80% (w/w) alcohol is prepared. Nine parts by weight of technical grade sodium nitrite is placed in a spray mixing bowl. The coating mixture is sprayed onto the 9 Received at IPONZ on 12 January 2011 C01.008NZ III technical grade sodium nitrite with the application of air heated to a temperature of approximately 50 °C. Flash evaporation of the solvent provides the MSN (90% (w/w) sodium nitrite).

Method 2 To a 10 L pail 4 Kg ethyl alcohol and 500 g water is added. To this volume 500 g zein is slowly added with continuous stirring to provide a 'zein solution'. The 'zein solution' is stood overnight and sieved before use. 8 Kg of sodium nitrite is 10 heated to 4 0 °C in a spray coating bowl and the temperature maintained during the spraying process. The total 'zein solution' is sprayed into the spray coating bowl and stirred until blended and dried. The coated sodium nitrite is passed through a coarse sieve, dried and the MSN (95% (w/w) sodium 15 nitrite) packed into plastic bags for storage prior to use.

PREPARATION OF BAIT-RITE™ To a mixer bowl 5.000 Kg of mixed grains and 8.400 Kg ground maize are added and stirred. The mixture is then blended with 22.384 Kg peanut butter followed by colourant (CAS# 2353-45-9). 20 To the 40 Kg batch of non-toxic paste composition 4 Kg of MSN is added and blended PIG STUDIES Initial experiments with baits containing technical grade sodium nitrite in cereal paste were unsuccessful as the taste of the 25 poison deterred pigs from eating bait. None of a group of six pigs presented with poisoned bait ate sufficient material to be killed. One animal, a pig which ate the most material, was severely affected, but recovered.

In a first trial the formulation was found to be palatable, 30 effective and humane, with thirteen out of fifteen pigs (87%) killed quickly. The two survivors did not eat a lethal dose, with one being a reluctant feeder, even of non-toxic material, and the second being sub-lethally dosed after spilling the bait C01.008NZ III Received at IPONZ on 12 January 2011 and the bolus of the toxin. When these two animals are excluded a 100% kill rate was achieved.

In a second trial data on the effect of omeprazole (a proton pump inhibitor) as a potential synergist was also gathered. 5 Omeprazole addition to the bait had a slight, but not significant, effect on the toxicity of microencapsulated sodium nitrite and the subsequent time to death.

The formulation of microencapsulated sodium nitrite presents a number of advantages. Firstly, the feeding deterrence observed 10 with formulations where sodium nitrite is distributed throughout a carrier material is avoided. Secondly, the need to include a proton pump inhibitor to reduce degradation of the toxic agent in the stomach is negated.

Trials For Study 1 twelve pigs were placed in two large pens and fed on a grain based diet with water ad libitum. Pigs were acclimatised for approximately one week before the trial began and were lightly fasted on the day before the trial. For Study 2 fifteen pigs were used with the same husbandry conditions.

Sodium nitrite was presented as technical grade sodium nitrite (NaN02) with a cereal paste bait 213 FERAEED™ (Connovation Limited) or microencapsulated sodium nitrite with a cereal paste bait designated 213 (Connovation Limited). Dose levels were chosen based on earlier work (Cowled et al, 2008) .

Study 1 - Technical grade sodium, nitrite The twelve pigs for this study were divided into four groups of three. Each pig received 150 gm of formulation. The following formulations were tested: 1. Technical grade sodium nitrite as a bolus in bait (bolus); 30 and 2. Technical grade sodium nitrite mixed throughout bait (mixed). 11 Received at IPONZ on 12 January 2011 C01.008NZ III There were two control groups and two test groups. The first control group received the bait alone and the second control group received the bait plus the carrier material. The first test group received technical grade sodium nitrite and carrier 5 material as a bolus in the bait. The second test group received technical grade sodium nitrite and carrier material mixed throughout the bait.

Bait consumption was assessed and compared with and without technical grade sodium nitrite and the effect of the poison 10 determined (Table 1).

Formulation Dosage mg/kg Number of pigs First control 0 3 Second control 0 3 Bolus 350 3 Mixed 350 3 Table 1. Summary of pig treatment with technical grade sodium nitrite including delivery method, dosage and number of pigs.

Study 2 - Microencapsulated sodium nitrite Microencapsulated sodium nitrite was prepared consisting of 80% (w/w) and 20% (w/w) encapsulating material.

The fifteen pigs for this study were divided into seven groups. Each pig received 150 gm of bait. The following formulations were tested: 1. Microencapsulated sodium nitrite as a bolus in bait (bolus); and 2 . Microencapsulated sodium nitrite mixed throughout bait (mixed).

No non-toxic control group was included, as most pigs 25 immediately ate the bait regardless of the presence of the toxin. 12 C01.008NZ III Received at IPONZ on 12 January 2011 Results The results from both trials are summarised below. In Study 1 sodium nitrite was ineffective.

Study 1 - Technical grade sodium nitrite Baits containing technical grade sodium nitrite were ineffective.

The lack of efficacy was attributed to taste deterring the pigs from eating the formulation (Table 3). The control bait without toxin was readily eaten. The carrier had a deterrent effect 10 which was further exacerbated by the presence of sodium nitrite when it was spread through the bait. None of a group of six pigs presented with a formulation comprising technical grade sodium nitrite was killed.

Formulation Dosage mg/kg Number of pigs* Mixed 300 3 Mixed 300 + omprazole 3 Mixed 350 2 Mixed 400 2 Bolus 215 1 Bolus 350 1 Bolus 400 1 Table 2. Summary of pig treatment with microencapsulated sodium nitrite including delivery method, dosage and number of pigs (* 2 pigs did not eat a full dose and are not included).

Study 2 - Microencapsulated sodium nitrite Baits containing microencapsulated sodium nitrite were palatable, effective and humane.

As indicated in Table 4 and in Figure 1 the results obtained with microencapsulated sodium nitrite as a bolus (2.6g pellets of compressed microencapsulated sodium nitrite) or mixed through 25 the bait both demonstrate linear decreases in time to death with increasing dose. 13 C01.008NZ III Received at IPONZ on 12 January 2011 The presentation as a bolus significantly reduced time to death in those animals receiving a lethal dose. However, delivery was more unreliable, with one pig partly spilling and leaving up to half the dose uneaten and surviving. This was not the case for 5 the presentation mixed through the bait which was consistently eaten.

Group Bait type Bait remaining (g) Amount eaten (g) Average eaten per group (g) 1 First control 0 170 170 0 170 0 170 2 Second control 47 123 74.3 107 63 113 57 3 Bolus 134 26 67.7 59 111 114 56 4 Mixed 154 16 28.7 113 57 157 13 Table 3. Bait consumption by individual pigs eating toxic and nontoxic bait.

The microencapsulated sodium nitrite was accepted by all test animals, with no aversion evident. The onset of symptoms was delayed for approximately 1 hour after the baits had been eaten. This was attributed to the delayed absorption of the active agent in the duodenum. Symptoms of methaemoglobinemia ensued 15 shortly thereafter, including loss of colour, lethargy and death.

Data on omeprazole as a potential synergist was also gathered in these trials. Omeprazole was administered at a dose of 40 mg, a dose known to increase gastric pH in pigs (Friendship et al, 20 2000) . Comparison of a dose of sodium nitrite with and without 14 C01.008NZ III Received at IPONZ on 12 January 2011 omeprazole showed that omeprazole slightly but not significantly reduced the time to death (One-way ANOVA; £"1,4=4.7; P=0.1).

Two results are not shown in Table 4 and Figure 1. A female that was fed a 350 mg/kg bolus dose only consumed half the pellets 5 (~170 mg/kg) and survived. A male, that would not eat the cereal paste bait, was alternatively fed 350 mg/kg with 150 g of pig pellets. Although this animal showed clear signs of toxicosis it also survived. It is, however, uncertain exactly what the final dose was that this animal received.

Dose (mg/kg) 300 300 350 400 215 350 400 Presentation mixed mixed + omeprazole mixed mixed bolus bolus bolus Individual times to death (min) 307 291 200 127 198 101 CO CO 293 232 200 132 340 272 Mean time to death (min) 313 265 200 130 198 101 CO CO SD 24 0 4 0 0 0 Table 4. Dose, type of delivery and individual and mean time to death for sodium nitrite in pigs In summary 13 out of 15 pigs (87%) were quickly killed. Two survivors did not eat a lethal dose. One was a reluctant feeder, 15 even of non-toxic material, and the second was sub-lethally dosed after spilling bait and a bolus of the toxin. When the reluctant feeder and the animal that split its dose are excluded a 100% kill rate was achieved.

The results suggest that microencapsulated sodium nitrite is a 20 humane and effective toxic agent for the control of feral pigs.

Received at IPONZ on 12 January 2011 C01.008NZ III Simulated Field Trial Materials Preparation of toxic bait A non-toxic cereal paste bait designated 213 FERAFEED™ 5 (Connovation Limited) containing microencapsulated sodium nitrite was prepared immediately prior to pen trials.

Microencapsulated sodium nitrite was determined by assay to comprise circa 80 % (w/w) sodium nitrite. Twenty five grammes (25 g) of the microencapsulated sodium nitrite was mixed with 10 225 g of 213 paste (FERAFEED™).

Pre-feeding and Exposure to Bait Station Nine domestic pigs (large white) weighing approximately 35 kg each were housed together in an enclosure with access to food and water ad libitum. Half of the enclosure could be closed to 15 form two smaller enclosures.

Two prototype bait stations were placed in one of the smaller enclosures. Six balls of bait weighing approximately 250 gms were placed in each bait station each day. The lids of the bait stations were wired open on the first night. The pigs were 20 allowed into the enclosure in groups of three and pigs fed readily from the bait stations.

On night two the bait station lids were closed. Pigs rapidly learned how to open the bait stations to access the non-toxic bait. Pre-feeding out of the bait stations was continued for 25 nine days.

Toxic Baiting Pigs were fasted for 24 hours and sorted into groups of three before entering the enclosure with the bait stations. Eight balls of toxic bait prepared as described above were placed in 30 the bait stations (four in each station) for Group One. Six 16 C01.008NZ III Received at IPONZ on 12 January 2011 balls of baits were placed in the bait stations (three in each station) for Groups Two and Three.

A veterinarian was present for the observation period.

Results Seven of the nine pigs consumed their food quickly and there appeared to be little aversion. Two of the pigs ate smaller amounts with one of the pigs being very timid and moving away from the bait when others approached.

Eight of the nine pigs died between 39 and 101 minutes. Average 10 time to onset of symptoms was 19.44 minutes, average duration of symptoms was 42.13 minutes and average time to death was 59.5 minutes. One pig survived.

All deaths were unremarkable and the following symptoms were observed in order: pale nose extremities, vomiting, blue 15 tongues, lethargy, ataxia, slight tremors, collapse and death.

The observations are summarised in Table 8.

Microencapsulated sodium nitrite was found to be an effective and humane poison for killing pigs in this study. Pigs easily learnt how to open the bait stations and fed readily on the 20 toxic baits.

Most pigs fed more quickly when feeding as a mob and challenged by others and one refrained from eating by moving away when challenged by other pigs. At least six of the pigs ate more than twice a lethal dose.

Times to death were relatively rapid compared with conventional vertebrate pesticides, but comparable with those reported for sodium nitrite in possums (Hix et al, 2010) and for other toxins inducing methaemaglobinaemia (Littin et al, 2010; Eason et al 2010a, b and c).

From the behavioural observations made after the pigs had consumed the toxin it seems possible that physical activity 17 Received at IPONZ on 12 January 2011 C01.008NZ III following bait consumption may enhance the toxic effects of the microencapsulated sodium nitrite.

Field trial Summary A trial was carried out in a fenced 20 acre block of Cypress-pine forest. A total of 12 feral pigs were captured, radio collared and released into the block of forest. Bait boxes specifically designed to exclude non-target species were set up at three sites in the block and monitored using infrared video 10 cameras to ensure individual pigs were accessing stations and that non-target species were excluded.

Each bait box was pre-fed with non toxic cereal paste bait for two nights and left empty on the third night. Pigs were observed opening the bait boxes on the first night they were 15 placed in the block and fed readily on baits. On the fourth night boxes were baited with toxic BAIT-RITE™ baits consisting of a microencapsulated formulation of sodium nitrite (MSN) mixed in a one to nine ratio with cereal paste bait.

On the first night of toxic baiting 11/12 pigs consumed a lethal 20 dose of the MSN paste bait and their carcasses were recovered, on average, 148 m from the nearest bait station, using radio tracking equipment the following day.

Observations made from the video cameras showed that pigs continued to consume baits well after they had consumed a lethal 25 dose. The microencapsulated formulation of sodium nitrite (MSN) presented in bait boxes was an effective method of killing pigs in a field setting while excluding non-target species.

Site The vegetation in the block consisted predominantly of several 30 species of the native Australian Cypress pine (Callitris genus). Twelve feral pigs (five males and seven females) were captured, 18 Received at IPONZ on 12 January 2011 C01.008NZ III radio collared, ear tagged and released at the trial site two weeks prior to the start of the trial.

Bait stations and pre-feeding All bait placement, toxic and non toxic, was carried out using 5 bait boxes. These consisted of plywood with a hinged lid and a stake behind the hinges that allowed lids to open but prevented them from staying open (Figure 3). Boxes were secured with two 4 0 cm metal stakes driven through holes in the base into the ground. Three bait boxes were used to bait pigs and these were 10 set up in a single area on the block. Boxes were placed approx ten metres apart in a triangle formation. Infrared motion detecting video cameras were set up on each of the three bait boxes and used to confirm pigs were accessing the boxes and that non-target species were excluded.

Boxes were baited with the non-toxic cereal paste bait shown to be palatable to pigs in earlier studies. Four baits, each weighing approx 250 g, were placed in each box on night one.

The same baiting was repeated on night two and then boxes were left empty on night three.

Toxic baiting On night four of the trial each box was baited with nine toxic baits making a total of 2 7 toxic MSN BAIT-RITE™ baits each weighing approx 250 g. Baits were prepared by mixing 25 g of MSN into 225g of cereal paste. MSN active was mixed into the 25 paste by flattening the paste and sprinkling the MSN active over the top. The paste was then rolled up and gently kneaded until the toxin was distributed throughout the paste.

Bait boxes and cameras were checked at sunrise the following morning. Baiting continued on night five and night six with two 30 250 g baits per box on each of these nights.

Pig carcasses were recovered using telemetry equipment. The location of each carcass was recorded using GPS and tissue samples were taken from each carcass. Tissue samples consisted 19 C01.008NZ III Received at IPONZ on 12 January 2011 of: stomach contents, stomach lining, muscle tissue taken from the left thigh, liver, kidney, large intestine and small intestine.

The stomach contents of individual pigs were also weighed and 5 all carcasses were disposed of by burning in a furnace. Rainfall and overnight temperature at the trial site was recorded for the five nights of non toxic and toxic baiting (Table 5).

Night Overnight temperature (°C) Rainfall (mm) 1 0 2 8 3 3 8 0 4 8 21 7 0 6 9 0 Table 5.

Non-toxic cereal paste and cereal paste containing MSN were prepared on site immediately prior to pre-feeding and the toxic section of the trial. Two samples of the MSN active were analysed to confirm the concentration of sodium nitrite (NaN02) active prior to the field trial. The two samples were found to 15 contain 95% (w/w) active. The assay was carried out according to the assay method for sodium nitrite taken from Analar Standards for Laboratory Chemicals (7th Edition).

Results On night four, the first night of toxic baiting, 11/12 pigs 20 consumed a lethal dose of the MSN BAIT-RITE™ paste bait. A total of 26/27 of the toxic baits were consumed and video camera footage showed all pigs consumed well in excess of a lethal dose.

On night four the only pig that did not consume any toxic bait 25 was observed as being excluded from the bait stations by other pigs. There was no bait take on night five or six and the one C01.008NZ III Received at IPONZ on 12 January 2011 surviving pig was seen to approach within five metres of bait stations, but did not access any of them. Bait was stopped after night six.

Pig carcasses were located on average 148 m from the nearest 5 bait station. The average individual weight of the stomach contents was 436 g. Pig measurements are provided in Table 6.

Pig tag # Sex Weight (kg) Stomach contents weight (g) Distance from bait stations (m) 40 F 33 134 155 186 M 182 184 M 32 186 216 43 F 469 139 187 M 46 170 275 41 F 722 84 44 F 33 CO CO 133 240 M 936 64 185 M 32 325 178 42 F 34 495 150 39 F 38 290 219 45 F 34 * * Table 6. *Pig did not access bait stations or consume any toxic bait Discussion The microencapsulated formulation of sodium nitrite (MSN) in cereal paste bait presented in bait boxes was an effective method of killing pigs in a field setting while excluding non-target species. Mob feeding mentality of pigs encouraged them to eat as quickly as possible and the bait box delivery method 15 further enhanced the effectiveness of the MSN bait.

Observations made from the video cameras showed that pigs continued to consume baits well after they had consumed a lethal dose. This observation supports the microencapsulation process masking the very salty taste of the SN active making the MSN 20 bait palatable enough for pigs to consume a lethal dose. 21 C01.008NZ III Received at IPONZ on 12 January 2011 POSSUM STUDIES Preliminary caged feeding studies provided a kill rate of 20 or 28 possums with doses of 50 to 750 mg/Kg indicating an LD50 around 120 mg/Kg. The purpose of the following trials was to 5 provide field efficacy data.

Field trials Summary A field trial site was established at Waikoko Farm near Little River on Banks Peninsula. The site was monitored prior to the 10 trials using wax tags and possum abundance was found to be 82.5% at the treatment site and 7 8.8% at the control site.

Pre-feeding at the treatment site was carried out using 213 paste placed in mini PHILPROOF™ bait stations, three times at one week intervals. Following this period of pre-feeding, 15 approximately 130 g of toxic bait was placed in each bait station. The toxic bait consisted of approx 10% MSN and 90% 213 paste. Baits were left out for four nights after which time post monitoring was carried out.

Post-monitoring found that possum abundance at the MSN treated 20 site had significantly reduced by 79.5% (p <0.01) to 16.25%.

There was no change in possum abundance at the control site (p = 0.48), with a post treatment abundance of 86.3%. Some possum carcasses were found within 5 to 10 metres of the bait stations.

Trial site The trial site for this study consisted of an area of land on the north face of a valley on Waikoko Farm near Little River on Banks Peninsula. The trial took place throughout June 2010, with an average temperature during this period of 6°C (min -3°C and max 14°C). Rainfall was 151% above normal (93 mm) whereas 30 sunshine hours were below normal (90 hrs). Weather data was sourced from the NIWA National Climate Centre. 22 Received at IPONZ on 12 January 2011 C01.008NZ III An area of approx 100 hectares of the face was used as a control (non-treatment) site and another area of approx 100 hectares was used as the treatment site. Vegetation consisted of open pasture, manuka (Leptospermum scoparium), totara (Podocarpus 5 totara), various Coprosma species, the native tree nettle ongaonga (Urtica ferox) and regenerating scrub-land.

Monitoring Possum abundance was measured pre- and post-poisoning at both treatment and control sites using the WAXTAG™ monitoring method 10 as described by the NPCA publication Possum population monitoring using the WAXTACP5 method (2008) . As per the protocol, lines consisted of 20 WAXTAG™ per line with 10 m intervals between tags. Four lines were placed in the control block and four lines in the treatment block. Pre-monitor lines were put 15 out when each site was established and tags were retrieved after seven nights as per the monitoring protocol.

The post-monitor of each site was carried out on the day that the toxic bait was removed from the site. The post-monitor involved placing out WAXTAG™ lines and again retrieving them 20 after seven nights to assess for possum chew marks.

Bait stations and pre-feeding A grid of 83 mini PHILPROOF™ bait stations was set out across the treatment site. Bait stations were set up at 100 m intervals on lines that were spaced 150 m apart in areas where vegetation 25 was sparse, and 100 m apart in areas of thicker scrub. Pre- feeding of bait stations involved the placement of approximately 200 g of non-toxic 213 paste in each mini PHILPROOF™ bait station. 213 paste is a non-toxic grain based paste bait commercially available and widely used by pest control 30 operators. Pre-feed baiting was carried out three times a week for three weeks before toxic baiting. 23 C01.008NZ III Received at IPONZ on 12 January 2011 Toxic baiting The toxic bait used in this trial consisted of MSN mixed into 213 paste, consisting of 10% MSN and 90% 213 paste. MSN was mixed into the 213 paste by flattening the paste and sprinkling 5 the MSN active over the top. The paste was then rolled up and gently kneaded until the toxin was distributed throughout the matrix.

Approx 130 g of toxic MSN paste bait was placed in each mini PHILPROOF™ bait station and toxic bait "take" was checked daily 10 following placement until day four when it was removed from each bait station. Bait was inspected in the bait station to ensure it remained in good condition.

Two samples of the MSN were analysed to confirm the concentration of silver nitrite prior to the field trial. The 15 two samples were found to contain 95% (w/w) silver nitrite active. The assay was carried out according to the assay method for sodium nitrite taken from Analar Standards for Laboratory Chemicals (7th Edition).

Results When bait stations were checked and cleared after four nights 74 of the 83 bait stations had experienced bait take, with nine stations being completely emptied. After four nights of toxic baiting field staff found seven possum carcasses in close proximity to bait stations. The seven carcasses were on average 25 12 m from bait stations with two possums within five metres of a bait station. There was a significant reduction (p <0.01, Figure 2) in possum abundance at the treatment site, with mean abundance decreasing by 7 9.5% (SE = 7.1%), from a mean BMI of 82.5% (SE = 11.1%) pre treatment to 16.2% (SE = 6.6%) post-30 treatment with SN. In contrast, there was no significant change in possum abundance over this period at the control site (p = 0.48), with a mean BMI pre treatment of 78.8% (SE = 7.8%) and post treatment of 86.3% (SE = 2.4%). 24 Received at IPONZ on 12 January 2011 C01.008NZ III The significant reduction in abundance at the treatment site occurred despite extremely wet weather conditions following toxin deployment and the baits appeared to be effective and able to withstand weathering effects for the duration of the trial. 5 Results were analysed using standard NPCA (2008) techniques for establishing stratified Bite Mark Indices (BMI) using wax tag data.

Discussion The significant reduction in possum abundance in the treatment 10 site of the trial shows that the MSN and 213 paste bait is both palatable and effective in a field setting. It also demonstrates that the encapsulation of the new formulation has a significant effect on efficacy. Earlier trials showed possums were very reluctant to eat baits containing unencapsulated sodium nitrite.

RAT STUDIES The efficacy of microencapsulated sodium nitrite (80% (w/w) sodium nitrite and 20% (w/w) encapsulating material) when used in the preparation of poisoned baits (213) was evaluated. Three groups of caged rats were permitted to feed on poisoned bait 20 containing microencapsulated sodium nitrite at ratios (w/w) of 1:10 (Group 1), 1:20 (Group 2) and 1:40 (Group 3). The results of these feeding trials are recorded in Table 7.

Effective (lethal) doses were ingested in the bait comprising 10% (w/w) sodium nitrite (NaN02) . The lethal dose (LD100) was 25 indicated to be in the range 170 to 410 mg/Kg. It was observed that tentative or prolonged feeders were less susceptible to ingestion of the poisoned bait.

Although the invention has been described by way of exemplary embodiments it should be appreciated that variations and 30 modifications may be made without departing from the scope of the invention.

Received at IPONZ on 12 January 2011 C01.008NZ III Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification.

In particular, although the invention has been described with 5 reference to the use of sodium nitrite as the toxic agent it will be recognised that formulations of other toxic salts of nitrite could be developed for use in accordance with the third aspect of the invention 26 C0L.CC3NZ III Received at IPONZ on 12 January 2011 Group SN % Rat Rat Quantity of bait ingested <g> Quantity of NaN02 ingested <mg) Rat weight (Kg) Dose (mg/kg) Death (D) or survival (S) 1 .0% IF 1 0.814 CO I-1 O 0.281 289.6797 D . 0% 2F 2 0 . 688 68 . 80 0 . 395 174 . 1772 D .0% 3M 3 1.577 157.70 0.506 311.6601 S .0% 4M 4 2 .754 275.40 0. 676 407.3964 D 2 .0% IF 0.981 49.05 0.258 190 .1163 D . 0% 2F 6 1.832 91. 60 0 .290 315.8621 S . 0% 3M 7 2.220 111.00 0 . 537 206.7039 S .0% 4M 8 2.209 110.45 0.476 232.0378 s 3 2.5% IF 9 11.796 294.90 0.329 896.3526 s 2.5% 2F 6.078 151.95 0.335 453.5821 s 2.5% 3M 11 12.122 303.05 0.258 1174.612 s 2.5% 4M 12 9.822 245 .55 0.273 899.4505 s 1 .0% SD, IS 2 . 0% .D, 3S 3 2 . 5% 4 S Table 7. 27 C31.000NZ III Received at IPONZ on 12 January 2011 Outcome Weight (Kg) Baits Total (g) Eaten (g) Time to onset of symptoms (mins) Time from first symptoms until death (mins) Time to death (mins) Group i (3 pigs) Survival - 36 -Death 32.0 8 x 250g 2, 000 670 25 68 93 Death 32.4 26 30 56 Group 2 (3 pigs) Death 35.4 10 32 42 Death 36.8 6 x 250g 1, 500 1, 500 15 39 54 Death 36.6 10 38 48 Group 3 (3 pigs) Death 26.4 25 76 101 Death 32.4 6 x 250g 1, 500 1, 500 14 29 43 Death 32.8 14 25 39 28 C01.008NZ III Received at IPONZ on 12 January 2011 REFERENCES Binkerd and Kolari (1975) The history and use of nitrate and nitrite in curing of meat Food and Cosmetic Toxicology, 13: 655-651.

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Claims (28)

1) A vertebrate pest control product comprising a microencapsulated alkali metal salt of nitrite. 5

2) The vertebrate pest control product of claim 1 where the alkali metal salt of nitrite is sodium nitrite.

3) The vertebrate pest control product of claim 1 or claim 2 where the ratio (w/w) of encapsulating material to alkali 10 metal salt of nitrite is in the range 0.025 to 0.5.

4) The vertebrate pest control product of claim 3 where the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.05 to 0.25. 15

5) The vertebrate pest control product of claim 4 where the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.05 to 0.07. 20

6) The vertebrate pest control product of any one of claims 1 to 5 where the encapsulating material is selected from the group consisting of: enteric coating material and lipophilic coating material. 25

7) The vertebrate pest control product of claim 6 where the encapsulating material is a water insoluble, digestible substance.

8) The vertebrate pest control product of claim 7 where the 30 water insoluble, digestible substance is zein.

9) The vertebrate pest control product of any one of claims 1 to 8 where the encapsulating material includes one or more organoleptic compounds. 35

10) The vertebrate pest control product of claim 9 where the encapsulating material includes one or more organoleptic compounds selected to promote the palatability to the 32 C01.008NZ III Received at IPONZ on 12 January 2011 vertebrate pest of the microencapsulated alkali metal salt of nitrite.

11) The vertebrate pest control product of any one of claims 1 5 to 10 where the vertebrate pest is selected from the group consisting of: feral pigs, possums and rats.

12) A vertebrate pest control product comprising microencapsulated sodium nitrite (MSN) where the 10 encapsulating material is zein at 5% (w/w) of the microencapsulated sodium nitrite.

13) A vertebrate pest control product comprising 10 % (w/w) MSN dispersed in a non-toxic paste composition. 15

14) Use of an effective amount of a microencapsulated alkali metal salt of nitrite in the preparation of a poisoned bait for the control of a vertebrate pest. 20

15) The use of claim 14 where the alkali metal salt of nitrite is sodium nitrite.

16) The use of claim 14 or claim 15 where the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is 25 in the range 0.025 to 0.5.

17) The use of claim 16 where the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.05 to 0.25. 30

18) The use of claim 17 where the ratio (w/w) of encapsulating material to alkali metal salt of nitrite is in the range 0.05 to 0.07. 35

19) The use of any one of claims 14 to 18 where the encapsulating material is selected from the group consisting of: enteric coating material and lipophilic coating material. 33 C01.008NZ III Received at IPONZ on 12 January 2011

20) The use of claim 19 where the encapsulating material is a water insoluble, digestible substance.

21) The use of claim 20 where the water insoluble, digestible 5 substance is zein.

22) The use of any one of claims 14 to 21 where the poisoned bait includes one or more organoleptic compounds. 10

23) The use of claim 22 where the poisoned bait includes one or more organoleptic compounds selected to promote the palatability to the vertebrate pest of the poisoned bait.

24) The use of any one of claims 14 to 23 where the vertebrate 15 pest is selected from the group consisting of: feral pigs, possums and rats.

25) The use of any one of claims 14 to 24 where the amount of microencapsulated alkali metal salt of nitrite used in the 20 preparation of the poisoned bait provides a concentration of the microencapsulated alkali metal salt of nitrite in the poisoned bait in the range 2.5 to 20% (w/w).

2 6) The use of claim 25 where the amount of microencapsulated 25 alkali metal salt of nitrite used in the preparation of the poisoned bait provides a concentration of the microencapsulated alkali metal salt of nitrite in the poisoned bait in the range 5 to 15% (w/w). 30

27) The use of claim 26 where the amount of microencapsulated alkali metal salt of nitrite used in the preparation of the poisoned bait provides a concentration of the microencapsulated alkali metal salt of nitrite in the poisoned bait in the range 7.5 to 12.5% (w/w). 35

28) A poisoned bait for the control of a vertebrate pest prepared by the use of any one of claims 14 to 27. 34 Received at IPONZ on 12 January 2011 C01.008NZ III 2 9) A microencapsulated alkali metal salt of nitrite manufactured for use in the preparation of the vertebrate pest control product of any one of claims 1 to 13. By our attorney Stephen R Parker, PhD 35