WO2000004780A1

WO2000004780A1 - Pesticide formulation

Info

Publication number: WO2000004780A1
Application number: PCT/AU1998/000581
Authority: WO
Inventors: Stephanie Smith
Original assignee: Bretts Pty. Limited
Priority date: 1998-07-22
Filing date: 1998-07-22
Publication date: 2000-02-03
Also published as: JP2002521314A; AU8425898A

Abstract

A pesticide which includes an extract from white Cypress pine (Callitris columellaris F. Muell.). The pesticide is prepared by extracting the Cypress pine with a solvent selected from an alkane, an alkoxyalkane or an alkylester. The solvent is then removed. Preferred solvents are n-hexane, diethylether and ethylacetate. The extracts can act as termiticides and are particularly effective against the subterranean termite C. acinaciformis.

Description

TITLE: PESTICIDE FORMULATION

THIS INVENTION relates to pesticides. In particular, it is directed, but not limited, to a

pesticidal composition for use in the protection of buildings, fences and other timber

constructions from attack by termites and similar pests.

Effective control of termites is a major problem throughout the world. Methods currently

employed to control these pests generally spray a liquid pesticide formulation onto the soil

before any foundations for constructions are laid, so that the water carriers, solvents,

emulsifiers, etc. dry out. the active pesticide remaining in the soil as a termite barrier.

However, the majority of these pesticides are synthetic and with the general public increasingly questioning the use of synthetic chemicals in the environment, governments

promulgate legislation which bans the use of such chemicals.

Accordingly, recent attempts to produce a more environment-friendly alternative include

the use of a physical rather than a chemical barrier to prevent the termite or other pest

species from reaching the area to be protected.

While these physical barriers may be an alternative, they are not as convenient to apply as

a chemical composition. For example, excavation around the perimeter of an existing

building is often necessary to apply a physical barrier, whereas a chemical composition can simply be sprayed around that same perimeter allowing the composition to seep into the soil around the building or possibly

directly into the timber to be protected.

Therefore, there remains a need for a chemical pesticide composition which is as

least similarly effective as the earlier synthetic compositions but without the

related environmental concerns.

Further, currently in the timber processing industry, there are large quantities of

sawdust which have to be disposed of, once again, according to strict government

regulations. A favoured method of disposal is by burning. Therefore, if at least

some of this sawdust could be further processed to provide a new and valuable

commercial product for the timber industry, this would also reduce the quantity

of sawdust to be disposed of by burning with a consequent reduction in running

costs for the timber mills.

Thus, any chemical which is naturally occurring and functions effectively as a

pesticide, particularly as a termiticide, and which can be manufactured from a

material which is currently simply disposed of would be of value.

It is a general object of the present invention to overcome, or at least ameliorate,

one or more of the above disadvantages. It has been discovered that the general object can be achieved using an extract

from Cypress pine, the extract containing components that are toxic or repellent,

or both, to termites and similar pests.

According to a first aspect of the present invention, there is provided a pesticide

comprising a Cypress pine extract, said extract prepared by:

a. extracting Cypress pine timber with a solvent that removes from the

Cypress pine at least those components which are toxic or

repellent, or both, to termites and similar pests; and

b. removing the solvent to obtain the extract.

Preferably, the Cypress pine is white Cypress pine (Callitris columellaris F.

Muell.)

Preferably, the solvent is selected from the group consisting of hydrocarbon,

substituted hydrocarbon and ester.

More preferably, the hydrocarbon is an alkane, the substituted hydrocarbon is an

alkoxyalkane and the ester is an alkylester. Most preferably, the alkane is n-hexane, the alkoxyalkane is diethylether and the

alkylester is ethylacetate.

Although not limited thereto, the Cypress pine extract of the present invention is

particularly effective against subterranean termites such as Coptotermes

acinaciformis.

Thus, according to a second aspect of the present invention, there is provided a

method for the control of pests, said method comprising the application of an

effective amount of a pesticide as hereinbefore described to an area requiring said

control.

Optionally, the extracts of the present invention may also include a carrier,

diluent or adjuvant.

As used throughout the specification, the term "carrier or diluent" denotes an

organic or inorganic, natural or synthetic material with which the aforementioned

extract is combined in order to facilitate the application of that extract to the area

to be treated. This carrier or diluent is generally inert. Similarly, the term

"adjuvant" has the usual meaning in the art to describe a material which aids the

operation of the extract. Preferred embodiments of the present invention will now be described with

reference to the following examples.

General extraction procedure

Callitris columellaris F. Muell. heartwood was steeped in the selected solvent for

24 hours at ambient temperature. The mixture was filtered and the filtrate

retained. The residue was steeped in a fresh sample of the selected solvent for a

further 24 hours at ambient temperature and again filtered. The filtrates were

combined and the solvent removed by evaporation to leave the extract.

Extracts A, B, C, D and E

Following the general procedure described above, five extracts (identified as A,

B, C, D and E) from C. columellaris heartwood were produced by extracting the

timber with ethylacetate, diethylether or n-hexane. The respective quantities of

heartwood used and amount of extract obtained are given in Table 1.

Table 1. C. columellaris heartwood extracts

Application of extracts to filter paper

The extracts A to E were diluted using their respective solvents to concentrations

of 100%, 50%, 25%, 12.5%, 1% and 0.5%.

The extract solutions were applied to filter paper (Whatman no. 540). The filter

paper was 90 mm diameter, and had an average mass of 0.6 g. Five replicate

filter papers were treated for each solution, by pipetting 0.8 ml extract solutions

onto each paper. They were treated by drippling over the filter paper. The

treated filter papers were placed in a fume cupboard for more than 24 hours to

dry.

Termite source

Orphaned workers and soldiers of C. acinaciformis were collected from natural

subterranean colonies and were transported to the laboratory on the same day.

They were used in bioassays within one week of collection. All the orphaned groups of termites used in the bioassays initially comprised workers (95%

including the third instar workers) and soldiers (5%).

River sand bioassay

Washed river sand (40 g) was placed in Petri dishes (90 mm diameter), and de-

ionised water added to achieve 80% moisture content. A single filter paper,

either treated with the extract solutions or untreated (solvent control and

untreated control), was placed onto the surface of the sand. One gram of C.

acinaciformis was added to each Petri dish. The bioassay was conducted in a

conditioned room maintained at 27 °C and 75% relative humidity.

This method of laboratory bioassay allows the simple assessment of filter paper

consumption and termite mortality.

Mound material bioassay

The same methods used for the washed river sand bioassay were used as for the

river sand assay except that the sand was replaced with C. acinaciformis mound

material. The moisture content was 80% in the mound material.

All five extracts from C. columellaris were toxic to C. acinaciformis at the

highest concentrations tested in the river sand bioassay (Table 2). Those termites

exposed to filter papers treated with 50% or 100% concentrations died within one o to two hours of exposure. Those termites exposed to filter papers treated with

12.5%o or 25% extractive concentrations died widiin one to two days.

For those termites exposed to filter papers treated with 0.5% or 1% extract

solutions, the hexane extracts (D and E) were most toxic to terrnites in both the

river sand (Table 3) and mound material (Table 4) bioassays. After two weeks,

there was 100% mortality, and hexane treated filter papers were either not

attacked or simply visited by termites. The diethylether extract (C) also

prevented C. acinaciformis from attacking the treated filter papers, although some

termites survived the duration of the bioassays. Only those filter papers treated

with the ethylacetate extracts (A and B) allowed termite nibbles or slight attack at

the 0.5% concentration. There also tended to be greater termite survival within

the Petri dishes containing ethylacetate extracts compared to the other extract

treated filter papers.

While the extractive treated filter papers displayed toxicity and repellency to

termites, the solvent controls and untreated controls were attacked (Tables 3 and

4). All filter paper controls were either moderated or slightly attacked, and most

termites survived the two week test period. The results suggest that the mound

material bioassay was more favourable to termite survival and feeding tiian the

river sand bioassay. Table 2. Mean percentage mortality of C. acinaciformis after exposure to filter papers impregnated with extracts from white cypress pine, after one week in a washed river sand bioassay.

Table 3. Mean percentage mortality and consumption rate by C. acinaciformis after exposure to filter papers impregnated with extracts from white cypress pine, after two weeks in a washed river sand bioassay.

Key: M = Percentage mortality C = Consumption rate MA = Moderate attack SA = Slight attack

NA = No attack TN = Tennite nibble TV = temπte visit Table 4. Means percentage mortality and consumption rate by C. acinaciformis after exposure to filter papers impregnated with extracts from white cypress pine, after two weeks in a mound material bioassay.

Key: M = Percentage mortality C = Consumption rate MA = Moderate attack SA = Slight attack NA = No attack TN = Termite nibble TV = termite visit

Evaluation of extracts when impregnated into small wood blocks

Preparation of test blocks

White cypress pine extracts were prepared by extracting white cypress pine with

one of three organic solvents, and the solvent then removed to provide the 100%

active ingredient or pure extract. Dilutions were made using the appropriate

solvent, ethyl acetate, diethyl ether or hexane, to achieve extract concentrations of

12800, 6400, 3200, 1600, 800 and 400 ppm. Permethrin (Perigen 500:470 g/kg

m/m) was used as the comparative preservative at similar active ingredient

concentration levels. Etliyl acetate, diethyl ether, hexane and deionised water were

used on their own to produce the solvent and water treated control blocks. The blocks of radiata pine (Pinus radiata D. Don) were 20 x 20 x 5 mm, with the

grain in the 5 mm direction. Weighed blocks were vacuum impregnated with the

various preservative solutions or solvents by weighing them down in beakers. The

treatment schedule was 30 min vacuum at 95 kPa, introduction of solution under

vacuum, release vacuum and allow blocks to soak for 30 min at atmospheric

pressure. The specimens were then removed from solution, blotted to surface dry,

and weighed to determine solution retentions. Immediately after weighing, blocks

were placed in plastic bags within a covered box (fish tank), which were gradually

opened, so that the solvent would evaporate slowly over two weeks, to reduce

redistribution of the active ingredients within the blocks.

The blocks were then either left unleached, or leached. Those blocks leached were

placed in jars of water in a shaking water bath at 35° C for five days, with water

changed daily. Leached and unleached blocks (including solvent and water

controls) were vacuum oven dried at 40 °C for five days, cooled in a desiccator and

weighed, to provide the pre-bioassay mass.

Termite source for bioassay

Workers and soldiers of C. acinaciformis were collected from natural sources,

transported to the laboratory and used in bioasssay within one week of collection. No-choice feeding bioassay of leached and unleached blocks.

Each test block was placed in a separate glass jar ( 100ml) containing C.

acinaciformis mound material (lOg) moistened to 80% moisture content. To each

jar was added 1.0 g of C. acinaciformis (approximately 300 individuals),

comprising 80-85% workers, 5-10% soldiers, and less then 5% nymphal forms.

While termites have no-choice of wood block, the mound material provides an

alternative food source that prolongs the viability of termites within each jar. The

glass jars were sealed with a vented lid. All treatments were replicated five times.

The experimental units were stored in an insectary at 26 °C and 70% R.H for eight

weeks. At the end of the bioassay, test specimens were cleaned and weighed after

vacuum oven drying to determine percentage mass loss. At the end of the

bioassay, termites in jars were recovered and counted to deteιτnine mortality.

Choice feeding bioassay of unleached blocks

This experiment used specimens similar to those in the no-choice bioassay, except

that only blocks with highest retentions were examined and all were unleached.

The test arena units were five perspex dishes 145 mm in diameter. Each test arena

contained five blocks treated with either ethyl acetate extract, diethyl ether extract,

hexane extract, permetlrrin or water. The blocks were positioned in random order

witiiin each arena. The space between each block was 25mm. Subterranean

termites were introduced to the test specimens from a vial containing mound material (80% moisture content) and five grams of C. acinaciformis connected to the

perspex dish via corrugated cardboard.

weeks. Inspections were made every third day. If all the termites within a unit died

before the end of the test period, the whole unit was withdrawn from the

experimental setup and the wood specimens removed and washed prior to re_¬

conditioning and re-weighing.

At the end of the eight week bioassay all wood blocks were removed, washed under

mnning water, vacuum-oven dried at 40 °C for five days and cooled in a desiccator

and weighed to determine mass loss. Visual estimates of termite mortality were

made throughout the test period and at the end of the eight week test.

RESULTS

No-choice bioassay

Mass loss data

Both the leached and unleached solvent (ethyl acetate, diethyl ether and hexane)

and water treated control blocks were all heavily attacked by C. acinaciformis and

suffered mean percentage mass losses of more than 45% (Table 5). hi this regard,

up to 5% mass loss is tolerated as some surface nibbling of blocks may be required

before a termiticide can kill all termites. All concentrations of permetlirin protected the P. radiata blocks from significant

termite attack, whether they were leached or unleached after treatment. All mean

mass losses were below 5% (Table 5).

Blocks treated with the ethyl acetate extract of white cypress pine performed better

than the other cypress pine extracts. All concentrations of extract in unleached

blocks reduced mass loss compared to the ethyl acetate solvent controls (Table 5).

The two highest concentrations of the ethyl acetate extract 0.64% and 1.28%

provided acceptable protection to unleached blocks as the mean mas losses were

4.8%o and 4.6%> respectively. However, when blocks were leached the level of

protection was reduced (Table 1). The 0.64%) concentration allowed 14.8% mean

mass loss and the 1.28% concentration allowed 12.5%o mean mass loss for leached

blocks.

The diethyl ether extract was less successful at protecting P. radiata from

significent teimite attack at the concentrations examined (Table 5). While mean

mass loss of diethyl ether extract treated blocks was less than for deithyl ether

solvent controls, mean mass losses for both leached and unleached blocks were still

about 20%) or more.

The hexane extract shows potential as a termiticide as unleached blocks treated

with die highest concentration ( 1.28%) had mean mass loss of just 6.6% (Table 5). However, protection was lower when blocks were leached and the lowest mean

mass loss achieved was 26-29% with the three higher concentrations (Table 5).

Mortality data

Termite mortality was relatively low in jars containing the water and solvent treated

controls with only 20% or fewer number of termites dying over the eight week test

period (Table 6). Permethrin caused complete (100%>) termite mortality when used

at all but the lowest concentration. All white cypress pine extract treatments caused

greater termite mortality than the controls, with mean percentage mortalities of 50%

or more. Unlike permethrin, only the one or two higher concentrations of each

extract caused complete mortality. Interestingly, the highest concentration of

diethyl ether extract caused 100% termite mortality (Table 6), even though

percentage mean mass loss was not much reduced (Table 5).

Choice bioassay

The results for the choice bioassay are shown in Table 7. Water treated control

blocks were heavily attacked with a mean percentage mass loss of 58.2%o.

Permethrin was very effective witii none of the five blocks being attacked

significently. Of the extracts, the ediyl acetate extract again proved to be most

effective, with a mean percentage mass loss of only 4.7%. The diethyl ether and

hexane extracts also reduced termite damage compared to the controls, although in

this trial, the mean percentage mass loss for blocks treated with the hexane extract (13.7%) was slightly more than for diethyl ether extract (7.4%). Unlike the no-

choice test, none of the termites survived beyond the eight week test period (100%

mortality). The white cypress pine extract treated specimens were less attractive to

feeding termites than the water treated blocks, as noted by the number of termites

seen on blocks throughout the bioassay period. Peπnethrin treated blocks were

most repellent to termites.

The results suggest that white cypress pine extracts have the potential to protect P.

radiata from termite attack. The most effective extract was ethyl acetate, providing

a high level of protection from C acinaciformis at the two highest concentrations in

unleached blocks. While the hexane extract failed to control termites, the reduction

in mass loss of unleached blocks in the no-choice test suggests that control could be

achieved with somewhat higher extract concentrations. The diethyl ether extract

provided least control in the no-choice test, although it showed some effectiveness

in the choice bioassay.

One of the standard procedures used in evaluating termiticides as wood

preservatives is to vacuum oven dry blocks before and after bioassay, to enable

calculation of mass loss. Vacuum oven diying also provides accelerated ageing that

approximates the changes that might occur to timber after prolonged service

conditions. However, it will be appreciated that such drying may also at least

partially remove any material that may be active against the termites. The potential mobile nature of some of the extractive components is suggested by the

much reduced efficacy of extracts when blocks were leached. These results suggest

that the white cypress pine extracts will be most suited to exposure conditions, such

as wall house framing, where timber is not subjected to leaching. Alternatively,

higher extract concentrations could be used if treated timbers were to be used in

exposed conditions.

Table 5. No-choice test results showing mean percentage mass loss of blocks after eight weeks exposure to C. acinaciformis. Mean of five replicates. Standard deviation in brackets.

*water treated control blocks

= concentrations not tested unL = unleached blocks. Leach = Leached blocks Table 6 No-choice test results showing estimate of C. acinaciformis mortality after eight weeks in laboratory bioassay. Mean of five replicates.

* water treated control blocks

- = concentrations not tested.

UnL = unleached blocks. Leach = Leached blocks.

TABLE 7. Choice bioassay results showing percentage mass loss of blocks after eight weeks exposure to C. acinaciformis. Mean of five replicates.

T.C. = Termite Choice: order of preference for blocks, from highest to lowest termite numbers on blocks (water, hexane, diethyl ether, ethyl acetate and peraiethrin).

Evaluation of extracts as soil treatments against a subterranean termite

Preparation of white cypress pine extracts

White cypress pine extracts were prepared by extracting white cypress pine with either ethyl acetate, diethyl ether or hexane and the solvent then removed to provide the 100% active ingredients or pure extracts.

Extracts were first diluted with the appropriate solvent to 5% (50,000ppm) concentrations and then further diluted to 3000, 6000, 12000 and 24000 ppm using deionised water. The resulting emulsions were shaken vigorously before sand treatments. Clilorpyrifos at 500 ppm concentration was used for comparison. Soil barrier treatment

Washed river sand (pH 7) that contained no organic matter and had a moisture content of 20-25% was used as the treatment substrate. Treatment solutions were applied to a 40mm thick bed of about 800 g moist sand in a lunch-box container (210 x 135 x 75mm). Sand was also treated with diethyl ether, ethyl acetate or hexane to provide solvent controls. A further control was sand treated with deionised water. Solutions were applied at a rate similar to that used in commercial practice (5 1/m²). The treated sand was placed in a fume cupboard to air dry for one week and then on the laboratory bench for a further week.

Five replicate sand samples were taken from each lunch-box of treated solvent- treated or water treated sands. Each sand sample was transferred to a clear plastic test cylinder. These cylinders (200 mm long, 20 mm wide) were filled with single treatments of the sand so that there were five replicates of each. Starting from the bottom of the cylinder and moving upwards, the first 40 mm of tube contained moist corrugated cardboard. Immediately above the cardboard was a 300 mm long plug of 8% agar, 20mm of untreated sand (dyed with 0.05% cotton blue), 80 mm of treated sand, 20mm of 8%> agar and a wooden bait block of Pinus radiata D. Don (10 mm thick).

Workers and soldiers of C. acinacifoιτnis were collected from natural sources and transported to the laboratory. These termites were used in bioassay within one to two days of collection.

One hundred termites were added to the corrugated cardboard within each test cylinder. The cylinders were mounted vertically with termites at the base. The experimental units were placed in a conditioned room at 27 °C and 75% relative humidity (R.H.). The deptii of tunnelling and level of termite mortality i each cylinder was assessed every second day over a two week period. RESULTS

Solvent and water controls

Termites were able to tunnel through the sands treated with either water, diethyl ether, ethyl acetate or hexane. Full penetration was achieved within 2 - 3 days and termites were able to attack the wood bait blocks at the top of the test cylinders (Tables 8-10). Termite mortalities in the control cylinders were low, ranging from just 10 to 20% (Tables 8-10).

Ethyl acetate extract

Termites were unable to penetrate completely (i.e., the 80 mm treated layers) any of the ethyl acetate extract treated sands (Table 8). While there was some tunnelling by the termites into the 3000 ppm treated sand (up to 25mm), the 6000 ppm treated sand (up to 16mm), and the 12000 ppm treated sand (up to 3mm), there was no penetration of the 24000 ppm treated sand. None of the termites were able to reach the wood bait blocks near the top of the test units. Most termites died within two weeks. For each ethyl acetate concentration, mean mortality was over 90% (Table 8).

Hexane extract

Termites were unable to penetrate complete (i.e., the 80 mm treated layers) any of the hexane extract treated sands (Table 9). However, there was some limited tunnelling into sand treated with each concentration. Termite tunnelling was up to 24mm for the 3000 ppm treated sand, 20 mm for the 6000 ppm treated sand, 12mm for the 12000 ppm treated sand, and 13 mm for the 24000 ppm treated sand. None of the termites were able to reach the wood bait blocks at the top of the test units. The mean mortality after two weeks was over 85% for each concentration of hexane (Table 9). Diethyl ether extract

Termites were unable to penetrate completely (i.e., the 80mm treated layers) any of the diethyl ether extract treated sands (Table 10). However, there was some limited tunnelling into sand treated with each concentration. Termite tunnelling was up to 35 mm for the 3000 ppm treated sand, 40 mm for the 6000 ppm treated sand, 22 mm for the 12000 ppm treated sand and 38 mm for the 24000 ppm treated sand. None of the termites were able to reach die wood bait blocks at the top of the test units. The mean termite mortality after two weeks was 75% or more for each concentration of diethyl ether (Table 10).

Chlorpyrifos

Although termites were able to penetrate all the bottom 8% agar layer (3 mm), and most of the 20 mm layer of untreated sand, none could penetrate the chlorpyrifos treated sand. The mean length of termite tunnelling through sand treated with 500 ppm chlorpyrifos was just 1.4mm (Table 10). Mean termite mortality was 96% (Table 10).

The experiment established that white cypress pine extracts have potential as soil termiticides. Extract concentrations of 3000-24000 ppm (0.3-2.4%) restricted or prevented termite tunnelling through treated sand and protected all wooden bait blocks from termite attack.

Table 8: Laboratory evaluation of ethyl acetate extract of white cypress pine as a soil chemical barrier against C. acinaciformis within two weeks of exposure.

Table 9: Laboratory evaluation of hexane extract of white cypress pine as a soil chemical barrier against C. acinaciformis within two weeks of exposure_.

Table 10: Laboratory evaluation of diethyl ether extract of white cypress pine as a soil chemical barrier against C. acinaciformis within two weeks of exposure_.

Chemical analysis of extracts

Gas chromatographic-mass spectrographic analyses were undertaken on each of the extracts and, although in no way limiting the present invention, the following compounds at least are believed to be components of each extract:

calaπ guaiol ne

Compound (1) Compound (2)

Compound (3)

The results demonstrate that the present invention should find use as an environment-friendly alternative to the use of synthetic chemical pesticides and which should be easier to apply to the area to be protected than a physical pesticide barrier.

Further, it is envisaged that at least some of the sawdust that is currently burnt by the timber processing industry could be fiirther processed to obtain the pesticide of the present invention thus, not only providing a new valuable commercial product for the timber industry, but also reducing the quantity of sawdust to be disposed of with a consequent reduction in rarrning costs for the timber mills and pollution of the environment. It will be appreciated that the above examples are illustrative only of the present invention and that modifications and alterations can be made thereto without departing from the inventive concept as defined in the following claims.

Claims

OCLAIMS

1. A pesticide which includes an extract from Cypress pine, said extract prepared bv:

a. extracting said Cypress pine with a solvent that removes from said Cypress

pine at least those components which are toxic or repellent, or both_{, t}o termites and similar pests: and

b. removing said solvent to obtain said extract.

2. A pesticide as defined in Claim 1, wherein said Cypress pine is white Cypress pine (Callitris columellaris F. Muell.)

3. A pesticide as defined in Claim 1 or Claim 2 wherein said solvent is selected from

a group consisting of hydrocarbon, substituted hydrocarbon and ester_.

4. A pesticide as defined in Claim 3, wherein said hydrocarbon is an alkane_, said

substituted hydrocarbon is an alkoxyalkane and said ester is an alkvlester_.

5. A pesticide as defined in Claim 3, wherein said alkane is n-hexane. said alkoxyalkane is diethylether and said alkylester is ethylace_tate_.

6. A pesticide as defined in any one of Claims I to 5 which further includes a carrier diluent or adjuvant.

7. A method for the control of pests, said method including the application of an

effective amount of a pesticide as defined in any one of Claims I to 6 to an area requiring said control.

8. A method as defined in Claim 7, wherein said pests are termites_.

9. A method as defined in Claim 8. wherein said termites are Coptotermes acinaciformis.