WO2018037093A1

WO2018037093A1 - A method and substrate with abamectin and fenpyroximate for killing mosquitoes

Info

Publication number: WO2018037093A1
Application number: PCT/EP2017/071376
Authority: WO
Inventors: Mikkel Vestergaard Frandsen; David Kim
Original assignee: Vestergaard Sa
Priority date: 2016-08-24
Filing date: 2017-08-24
Publication date: 2018-03-01

Abstract

Abamectin and Fenpyroximate are provided on a non-living substrate for killing mos- quitoes. For example, Abamectin and Fenpyroximateare incorporated in a thermo- plastic substrate for migration to the surface from which the agents are transferred in combination to the mosquito in order to act synergistically.

Description

A method and substrate with Abamectin and Fenpyroximate for killing mosquitoes

FIELD OF THE INVENTION

The present invention relates to use of an insecticidal combination of Abamectin and a Fenpyroximate. Specifically, it relates to a method for killing mosquitoes. It also relates to a substrate for such killing action.

BACKGROUND OF THE INVENTION In the fight against malaria, there is a steady trend towards new pesticide combinations, including the aim of fighting pesticidal resistance among mosquitoes and in order to increase the efficacy. Accordingly, there is an on-going search for combinations of pesticidal agents that exhibit a synergistic effect, meaning that the combination of the active ingredients has a higher efficacy than the mere sum of the efficacies of the active ingredients. For example, if one of the ingredients has no effect, and the other ingredient has a moderate effect, a synergistic effect is proven for the combination of the pesticidal agents if the efficacy is higher than this moderate efficacy when the pesticidal agents are combined. Such synergistic effect has the advantage of reducing the necessary amounts and concentrations of the insecticides, which is a general desire, and it can give a good measure to kill insects despite insecticidal resistance against specific insecticides or even classes of insecticides, as well as cross-resistance. A model of describing synergistic effects is found in the article "An overview of Drug Combination Analysis with Isobolograms" by R.J.Tallida, published in The Journal of Pharmacology and Experimental Therapeutics, Vol. 319, No. 1, 2006, pagesl-7.

As an alternative to synergistic effect, new insecticides are proposed constantly, as well as new combinations of insecticides with additive effects or where one insecticide takes the role of the killing when the other is not efficient due to insecticidal resistance by the insect. In the article, "Susceptibility of Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say to 19 Pesticides with Different Modes of Action" published by Pridgeon et al. in J. Med. Entomol. 45(l):82-87 (2008), found that Abamectin had a multi-fold lower efficacy on mosquitoes as compared to Permethrin, in particular a 9-fold lower efficacy for the mosquito strain Ae aegypti. The mortality was determined 24 and 36 hours after exposure of the mosquito to the pesticide.

When having regard to the results in the article by Pridgeon of Abamectin having a killing efficacy of an order of magnitude lower than Permethrin, one could speculate whether this is the main reason why Abamectin has not found way to the market for fighting mosquitoes. However, there is another very important reason that is important to notice. Although, Abamectin has a killing effect on mosquitoes in general, the reaction is very slow. Thus, no quick knock down is achieved after exposure. This very important disadvantage of Abamectin in relation to killing mosquitoes is not reflected in the in the article by Pridgeon, as the mortality was only measured after 24 or 36 hours. Whereas, when used as a miticide in agriculture, a slow knock-down of mites is acceptable, if just the killing is efficient, the requirements for preventing malaria are very different. In this case, a quick knock down is essential. If the knock-down is slow as for the Abamectin, the mosquito can still bite after exposure to Abamectin, which is not useful in malaria prevention.

Accordingly, mectins are mostly known as efficient miticides. For this purpose, mectins are reported to have enhanced efficacy when combined with certain mitochondrial elec- tron transport inhibitor (METI). Mectins in general include Abamectin, Emamectin (typically provided in the salt form Emamectin benzoate), Lepimectin, Milbemectin, and Ivermectin. Mitochondrial electron transport inhibitors (METI) in general include Fenazaquin, Fenpyroximate, Pyrimidifen, Pyridaben, Tebufenpyrad, Tolfenpyrad. In particular, Abamectin is a pesticidal neurotoxin, widely used for protection of agricultural plants against pests, especially mites. Within the last two decades, a large number of combinations of Abamectin with other insecticides have been disclosed. A combination of Abamectin with a metabolic inhibitor against mites and insects in agriculture is described in the Chinese patent applications CNlOl 176457 by Dongguan Ruidefeng Biotechnology Co Ltd, disclosing synergistic mixtures of Abamectin with Fenpyroximate for crop protection. Specified insects are spider mites, moths and leafminers. CN103636634 by Gaungxi Tianyuan Biochecmistry Co discloses Fenpyroximate in combination with various active ingredients against mites, wherein Avermectin is one option for such active ingredient. Exemplified are 0.5% Fenpyroximate with 3% Avermectin and 2% Fenpyroximate with 1.5% Avermectin. Accordingly, synergistic effects were observed in cases where there was more or less Fenpyroximate than Abamectin. Efficacy results of insecticidal combinations that work on acari do not necessarily work on insects, and concentrations that have high efficacy on acari do not necessarily yield good results for insects. Furthermore, a high insecticidal efficacy of insecticides on one species of insects does not necessarily yield likewise results for other insects. In addition, insecticidal efficacy may vary for an insect species in dependency of the various stages of the life cycle. For example, some insecticides and combinations of insecticides may work on mosquito larvae and not on adult mosquitoes. This is experimentally verified in the article "Evaluation of Novel Insecticides for Control f Dengue Vector Aedes eagypti (Diptera: Culicidae)", by Paul et al, published in in J. Med. Entomol. 43(1): 55- 60 (2006). It reads in the abstract that "Z¾e effect of PBO on the toxicity in adults and larvae was considerably different, both in terms of the insecticide synergized (or antagonized for chlorfenapyr versus adults) and in terms of degree of synergism" .

Thus, finding useful combinations of insecticides in order to kill adult mosquitoes is a generally difficult task, especially because the insecticides should also be acceptable in connection with use in dwellings.

Whereas, in agriculture, a typical way of applying pesticide combination is by spraying or adding pesticide carriers to the plants and soil, in dwellings, apart from spraying internal walls, often, the insecticide is provided on a substrate, for example a textile, such as a mosquito net. Thus, not only are different insecticides employed but also different ways of exposing the insects to the insecticides. Therefore, acaricidal science in agriculture and human protection against mosquitoes are typically very different fields with a low degree of overlap with respect of how insecticides are to be used and in which combinations and at what concentrations.

In order for pesticides to act in combination, they typically should be applied together or at least subsequently within a short time interval. Various methods are discussed in the literature, for example in order to fight mosquitoes or other insects. Substrates with various combinations of pesticidal agents are many-fold and include

- a plurality agents distributed evenly in a coating on the substrate, for example as disclosed in WOO 1/37662;

- two agents evenly incorporated into a polymer of a substrate with migration to the surface, for example as disclosed in WO2010/015257; differential migration control with different migration inhibitors inside the matrix is discussed in WO2003/063587, although in WO2009/003468, it is pointed out that different migration inhibitors influence each other such that a proper control is very difficult;

- two agents incorporated in different parts of a fibre, for example as disclosed in WO2009/003468, such as in the core and shell of a bi-component fibre;

- two agents incorporated in different filaments combined into a single yarn, for example as disclosed in WO2009/003468 or WO2011/124228;

- two agents in different yarns combined into a textile, for example as disclosed in WO2010/046348.

For regulation of migration of pesticidal agents in polymers, various methods have been proposed, although, it is not always agreement to the effect of these means. For example, ethylene vinyl acetate (EVA) has been proposed in WO2003/063587 for promoting migration, whereas US6979455 explains that EVA retards migration. With respect to clay in the role of a migration controlling agent, kaolin has been proposed for increasing migration in WO2003/063587, whereas US3859121 proposes clay for retarding migration. For other migration controlling agents, similar discrepancies are found in the art. Thus, there is apparently no clear agreement in the art with respect to the effect of migration control.

The large variety of possibilities for apparently controlled release and the missing agreement on the effect of migration controllers reflect the desire in the art to find good solutions for differential migration control for two or more different agents in a polymer substrate. No clear trend is recognizable in the field other than a steady increase of proposals of various kinds. Thus, there is still a desire in the art for good solutions in differential migration control.

DESCRIPTION / SUMMARY OF THE INVENTION

It is an objective of the invention to provide an improvement in the art. Especially, it is an objective to provide a novel, useful combination of pesticidal agents against mosquitoes. It is also an objective to control the release of the combination of pesticides. These objectives are achieved by using a combination of Abamectin and Fenpyroximate as explained in detail in the following. For Abamectin, as already described above, it has been observed that there is only a slow knock down of mosquitoes such that it, despite a reasonable killing effect, does not appear useful in fighting malaria, where quick knock-down is essential to prevent spreading of the disease. On the other hand, it has been observed experimentally that Fenpyroximate has a relatively quick knock-down efficacy against mosquitoes but, unfortunately, a low killing effect, as the mosquitoes appear to recover again after hours or days. From this perspective, Fenpyroximate appears also not as a useful candidate in the fight of malaria.

However, it has been found in experiments, that the efficient and quick knock down efficacy of Fenpyroximate can be successfully combined with the efficient, delayed killing effect of Abamectin. Before the mosquitoes can fully recover from the knock-down by Fenpyroximate, the Abamectin develops a lethal effect inside the mosquito. For this reason, two apparently useless insecticides for fighting malaria turn into potent pesticides when used in combination.

In the fight against malaria, it is important to provide long lasting insecticidal barriers, such as mosquito nets or wall coverings, also called wall linings. However, providing such long lasting barriers that last for several years is very difficult, as the insecticides tend to be exhausted quickly. For insecticidal combinations there is an added difficulty in that one insecticide tends to be exhausted before the other. This is especially the case when the barrier is washed repeatedly by which the insecticides are removed from the surface to a large extend. When combinations of two insecticides are incorporated into a bulk polymer for migration to the surface, the washed-off insecticides are replenished from the bulk due to a concentration gradient between the bulk and the surface. This replenishing gradually reduces the concentration in the bulk. If, then, one insecticide is washed more off the surface than the other, for example due to higher water solubility, the concentration ratio is changed, and one agent is lost faster than the other over time. Other effects, such as chalking and different ease of abrasion may add to one of the insecticides being exhausted faster than the other, which may reduce the overall insecticidal lifetime of the barrier.

In the examples with Abamectin and Fenpyroximate, it has been found experimentally that Abamectin tend to be lost faster than Fenpyroximate. However, surprisingly, it has turned out that this does not reduced the lifetime of the product substantially, if some special precautions are taken as explained in the following.

It has been verified experimentally that the concentration ratios between Abamectin and Fenpyroximate influence the efficacy. For some mosquito strains, varying the concentration ratio changed an additive lethal effect into a synergistic or antagonistic effect. The latter is an interesting effect which can be utilised in a non-traditional way for achieving a long term insecticidal efficacy despite loss of one agent faster than the other. For example, an extended long term efficacy is obtained by providing a substrate, for example a mosquito net, with an initial concentrations of Abamectin and Fenpyroximate that result in an additive effect. At the initial state of the substrate, the additive effect is sufficient for efficient killing, as the concentrations are also relatively high. With time, the concentrations go down and will tend to reduce the efficacy with time. However, the faster reduction of the concentration of Abamectin causes a change in the concentration ratio from the additive regime into a synergistic regime. The synergistic effect, then, compensates for the general decrease in efficacy due to reduced surface concentration on the substrate. Thereby, a prolonged insecticidal efficacy is obtained despite reduction of insecticide concentration. As it appears from the considerations above, the use of Abamectin and Fenpyroximate against mosquitoes, especially insecticidal resistant mosquitoes, is not a straightforward solution, as both Abamectin and Fenpyroximate appear useless in the fight of malaria at first sight and imply complex problems when used against mosquitoes. This is very different from the considerations that apply when using these two insecticides in agriculture against mites, where quick knock-down is not a necessity. However, by carefully studying the combined effect of Abamectin and Fenpyroximate against mosquitoes, the combination turns out to be useful when concentrations are adjusted to non- antagonistic regimes.

Specific synergistic or additive ratios of Abamectin relatively to Fenpyroximate have been found experimentally for various mosquito strains. For the mosquito strain Anopheles quadrimaculatus multi-fold more Fenpyroximate than Abamectin is neces- sary for not having an antagonistic effect. For some ratios, Abamectin and Fenpyroximate were found to act additively and for others even synergistically. An antagonistic effect should be avoided, which appears to be the case if a lower concentration is used for Abamectin than for Fenpyroximate. Although, the combination of Abamectin and Fenpyroximate against mosquitoes can be used in multiple ways, for example indoor or spraying, focus is given in the following to insecticidal barriers. Examples of such barriers are textiles, especially mosquito nets, also when used as screens for windows or doors. Other useful applications of such substrates are as wall linings, or coverings of ceilings and floors, such as carpets. How- ever, the substrate can also be a foil or a tarpaulin, optionally used as floor, ceiling, and/or wall of a dwelling, including tents, or as a curtain. Another use is an outdoor fence around open air areas, for example around dwellings, schools, playgrounds, yards, cattle fields, or animal stocks. The list of examples is not exhaustive. In the following, various embodiments are described in more detail.

Advantageously, the method comprises providing a non-living substrate, such as a mosquito net or a wall lining, having Abamectin as a first pesticidal agent and Fenpy- roximate as a second pesticidal agent on a surface region of the substrate and exposing the mosquito to the combination of the Abamectin and Fenpyroximate by contact of the mosquito with the surface region. For example, the substrate is provided with a coating or impregnation that contains the Abamectin and Fenpyroximate. Primarily, non-living substrates are envisaged, especially thermoplastic polymers. Alternatively, the substrate consists of or comprises a thermoplastic polymer having incorporated therein the Abamectin and Fenpyroximate for migration from a bulk of the substrate to the surface of the substrate. In any case, when contacting the surface of the substrate, the mosquito is exposed to the Abamectin and Fenpyroximate in combination, such that the combination can act synergistically or additively on the mosquito and not in the antagonistic regime.

The ratios for which an antagonistic effect is observed or an additive or synergistic effect varies in dependence of the strain of mosquitoes. However, useful surface concentration ratios have been found in the range of 1 : 1000 to 1 :3. For a precise determination of the most efficient ratio, a specific strain of mosquitoes is selected and a concentration ratio is experimentally determined for the specific strain, where the ratio exhibits additive or synergistic killing effect on the mosquito strain. The surface con- centration ratio is then adjusted for additive and synergistic killing effect. In those cases, where a specific mosquito strain is targeted, it is worth the effort and the corresponding adjustment. However, in many situations, it is sufficient to provide the surface concentration ratio such that the effect is not antagonistic. The additive killing effect is often sufficient to cover several mosquito strains and may with time due to concentration changes turn into a synergistic effect. For example, gradual change of the surface concentration ratio may be caused by faster exhaustion of Abamectin relatively to Fenpyroximate, changing the additive effect into the synergistic regime.

As an example, for some mosquito strains, a concentration ratio of larger than 1 :3 be- tween Abamectin and Fenpyroximate behaves antagonistic and lower than 1 :3 has an additive effect, while a concentration of less than 1 : 10 yields a synergistic effect. Providing Abamectin and Fenpyroximate at a concentration ratio of 1 :3 initially may yield a satisfactory effect, especially at high concentrations. The overall concentration of both agents will decrease due to gradual exhaustion of the Abamectin and Fenpyrox- imate in the bulk. In this connection, time frames of months and years are envisaged, thus, it is not a short term effect. If the concentration of the Abamectin falls faster than the concentration of the Fenpyroximate, the ratio between the Abamectin and Fenpy- roximate decreases correspondingly. The decrease of this ratio drives the efficacy into the synergistic regime if the ratio becomes less than 1 : 10, for example between 1 : 1000 and 1 : 10.

In some embodiments, the method comprises melt-incorporating the Abamectin and Fenpyroximate in the thermoplastic polymer of the substrate, extruding or moulding the thermoplastic polymer with the incorporated Abamectin and Fenpyroximate as part of the production of the substrate; causing the Abamectin and Fenpyroximate to migrate to a surface region of the substrate for providing amounts and weight ratios of the Abamectin and Fenpyroximate that act lethal, for example synergistically or addi- tively but not antagonistically, on the insect when contacting the surface of the substrate. Typically, only a single contact is required between the insect and the substrate for exposing the insect to the combination; this implies that the combination of both insecticidal agents is available on an area of the surface, the size of which is corresponding to the size of the insect. This requirement does not necessarily imply that the combination of Abamectin and Fenpyroximate is a mixture, as the two active agents can be provided from different filaments that are interwoven, knitted, or otherwise combined, for example twisted into a yarn or part of a nonwoven material.

Non-limiting examples of useful thermoplastic polymers for the substrate are polyester for coating, and polyethylene or polypropylene for incorporation. However, other thermoplastic polymers can also be used, especially other polyolefins or blends of poly- olefins,

For biocidal efficacy, the ratio by weight between the Abamectin and Fenpyroximate, to which the insects are exposed, is advantageously less than 1 :3, such that less Abamectin is used as compared to Fenpyroximate, in term of weight in a formulation or in the material of a substrate from which the agents migrate from the bulk onto the surface. The ratio may be dependent on the specific strain of mosquito. In experiments, it was found that a specific concentration of Abamectin had high killing efficacy on a VKPR strain of mosquitoes, whereas the same specific concentration had no killing efficacy on mosquitoes from Tiassale. However, surprisingly for the Tiassale strain, the killing efficacy for Fenpyroximate was tripled from 5.3% to 15.8% by adding such specific concentration of Abamectin. Thus, there was shown a clear synergistic effect with respect to mortality.

Useful concentration ratios for Abamectin and Fenpyroximate are between 1 : 1000 and 1 :3. Concentration ratios and concentrations are by weight.

These ratios are useful for concentrations of the pesticidal agents on the surface of the substrate. However, if the active agents are incorporated into a thermoplastic polymer for migration to the surface, the above stated ratios are also indicative of useful con- centrations ratios inside the bulk of the polymer. This is so because the bulk concentration ratio and the surface concentration ratio, typically, are in equilibrium. Therefore, to a good approximation, the bulk ratios are also useful in the range of 1 : 1000 to 1 :3.

In some embodiments, the substrate comprises a concentration of incorporated Abamectin in the interval of 0.25-25g per kg thermoplastic polymer of the substrate, for example in the interval of 0.5-5g/kg, and a concentration of incorporated Fenpyroximate in the interval of 1-100 g per kg of thermoplastic polymer of the substrate, for example in the interval of l-50g/kg, optionally l-25g/kg or 1-10 g/kg. The interval of 1-25 g/kg is in some cases preferable for minimising influence on the stability proper- ties when incorporated in yarns and extruded.

A synergistic effect is desirable due to an enhanced effect with reduced amounts of active agents. However, with respect to Abamectin and Fenpyroximate, also the additive effect has turned out to be utmost useful, as Fenpyroximate was observed to cause a fast knockdown whereas Abamectin acted slow but with high lethal efficacy. Thus, the two agents act complementary with respect to immobilizing the mosquitoes, where Fenpyroximate immobilizes on a short term but not necessarily kills the mosquito, and where Abamectin has a slow knockdown efficacy but immobilizes on the long term by causing death to the mosquito.

In more detail, it appeared from experimental studies on mosquitoes that the Fenpyrox- imate induced a fast and efficient knockdown (defined as early time point knock down, i.e., at 1 and 6 hrs) of the insects but did not efficiently kill (i.e., knockdown at 24 hrs) the mosquitoes; knockdown values were an increasing 40% already after 1 hour but did not reach more than 80% after 24 hours, which could be due to a part recovery of the mosquitoes. In contrast thereto, Abamectin had no knockdown efficacy within the first hour but turned out to be a potent insecticide with good mortality of 60% after 6 hours and 100% after 24 hrs, when using an exposure of 50 ppm. Thus, only the combination of Fenpyroximate and Abamectin yields a quick knockdown and a high mortality. When incorporated into a thermoplastic substrate, the Abamectin and Fenpyroximate are added to the molten polymer and then formed into the solidified substrate, typically by extrusion or moulding. For this reason, also the term "melt-incorporating" is used. The Abamectin and Fenpyroximate migrate to the surface of the substrate driven by the gradient between the bulk and the surface until equilibrium is achieved. In some em- bodiments, the substrate is a single type of thermoplastic polymer, for example a polyester or a polyolefin, such as polyethylene or polypropylene, or mixture of thermoplastic polymers.

In other embodiments, the substrate comprises different types of polymers that are not mixed but combined in other ways, for example by lamination, surface fusing, or by weaving or knitting or otherwise combining different types of fibres.

The two types of polymer can be identical or different, the difference being due to the ingredients in the polymer itself and/or by the polymer structure. For example, both types of polymer can be polyethylene, but one polyethylene may have different further ingredients as compared to the other, or the two polyethylene polymers may differ in their internal polymer structure, such as high density polyethylene versus low density polyethylene. Also, the different types of polymer can be different types of polyolefin. Optionally, the Fenpyroximate is loaded onto a support of micro-sized or sub-micron sized support particles, for example clay particles. For example, the size of the clay particles is around one micrometre or in the sub-micrometre range, which is sometimes called nanoclay. The support particles are then incorporated into the molten polymer prior to extrusion or moulding. Supports particles act as a reservoir for the Fenpyroximate and in some cases also as a retarding factor, which is useful for adjusting the long term release of Fenpyroximate. Optionally, at least two types of filaments are provided, one type comprising Abamectin, optionally not Fenpyroximate, and the other comprising Fenpyroximate, optionally not Abamectin. The different types of filaments can be combined as yarns into a woven, knitted or non-woven textile. Alternatively, different types of filaments can be intertwined into a single yarn. The term filament is used as single filament or multifilament, wherein the term multifilament is used if the filaments in the multifilament are identical. The term yarn also includes an assembly of different types of filaments. For the term yarn or filament, we will use the generalised term fibre. Typical fibre diameters are 0.05 to 0.35 mm. For example, the Abamectin is incorporated in a first type of thermoplastic polymer and extruded and formed into a first type of fibres, and the Fenpyroximate is incorporated into a second type of thermoplastic polymer and extruded and formed into a second type of fibres. The term "formed" is here used for the treatment after extrusion, for example stretching, twisting and possibly combining multiple filaments that are extrud- ed, for example by a multifilament spinneret. For example, the Abamectin but not the Fenpyroximate is incorporated in the first type of fibres, and the Fenpyroximate but not the Abamectin is incorporated in the second type of fibres.

These two types of fibres, and potentially further types of fibres, are then combined into a textile that forms the substrate or part of the substrate, for example a region of a substrate, optionally a region on one side of the substrate, for example only one side of the substrate. The principles can be used for more than two active ingredients in more than two types of fibres. For example, the fibres are then woven or knitted together to form the textile.

Alternatively, the fibres are combined as a nonwoven textile, for example by wetlay techniques, spunbonding, or needle punching. In this regard, it has been observed that insects, especially mosquitoes, have a higher likelihood to contact a large number of fibres in non- woven materials as compared to woven products. In order to provide a good stability of the non-woven textile, heat bonding binder fibres can be added to the textile. Such binder fibres have a lower melting point that the first and second type of the thermoplastic fibres in order to bind the non- woven into a stable structure at a relatively low temperature, minimizing the detrimental effect of high temperature on the active ingredients. Typically, the concentration of binding fibres is 1%-10% of the total weight of fibres in the non-woven textile substrate. For example, the heat melting binder fibres have melting point of between 80 and 120 degrees centigrade. If the first and the second type of fibres are made of a polyolefin, for example polyethylene or polypropylene, for which the melting temperature is above 130 degrees centigrade, the use of binder fibres is advantageous.

As discussed earlier, it is advantageous to delay the exhaustion of the Fenpyroximate relatively to the Abamectin in order to move from an additive into a synergistic regime. One possibility for obtaining such delaying effect is loading the Fenpyroximate onto support particles. The support particles are then incorporated into the common polymer of the substrate or, alternatively, into the second type of thermoplastic polymer prior to extrusion and formation into the second type of fibres.

In order to have a delayed exhaustion of the Fenpyroximate but, on the other hand not provide a delayed start concentration on the surface of the substrate, the method comprises in some embodiments dividing an amount of Fenpyroximate in to a first portion and a second portion, the first portion being 10%-90% or 20-80% or 30-70% or 40- 90% or 50-95% of the amount of Fenpyroximate, loading only the first portion on support particles and incorporating the support particles loaded with the first portion as well as the second portion of Fenpyroximate into the molten thermoplastic polymer, for example into the polymer for the second type of fibres, prior to extrusion. Typically, the non-loaded Fenpyroximate is almost instantly available on the surface, while the loaded Fenpyroximate is retarded even in case of a tendency of high abrasion or otherwise loss from the surface of the Fenpyroximate. Also, the loading prevents early exhaustion of Fenpyroximate due to incompatibilities between Fenpyroximate and the polymer matrix.

A typically used support particle type is clay, especially nano-clay. Examples of nano- clays are attapulgite and montmorillonite. As alternative to clay, other micro-particles or nano-particles are used, for example ground natural minerals or synthetic material, including silica, alumina and silicates. Examples of support particles include kaolin, talc, chalk, quartz, carbon black, diatomaceous earth, calcite, marble, pumice, sepiolite and dolomite. Useful types of support micro-particles with submicron pore sizes are disclosed in various patents by Amcol International Corporation and Amcol Health and Beauty Solutions Inc. For example, the weight percentage of the support is between half and two times of the weight percentage of the Fenpyroximate.

When using two type of fibres, the concentration of the Abamectin and Fenpyroximate can be regulated in the final product by adjusting the loading in the two types of fibres as well as adjusting the weight of the first type of fibres in the substrate as compared to the weight of the second type of fibres in the substrate.

For example, such mixture of the first and the second type of fibres are used for knitting or weaving a textile. Optionally, the first and second type of fibres are monofilament fibres, although multifilaments are also possible for such types of fibres. For example, the thermoplastic polymer is a polyethylene or polypropylene, although blends of polymers can be used as well. Alternatively, mixtures with such ratios between the first and the second type of fibres are used for non-woven textiles. Optionally, these two types of fibres are used in a nonwoven wetlay, spunbond or needle punch process for a textile. For the final substrate, the weight percentages of the first and the second type of fibres are adjusted to multi-fold less total Abamectin than total Fenpyroximate in the final product, for example at least 3 times less.

In some cases, such as explained in WO2010/046348, the ratio between the concentra- tions of two agents on the surface is desired to be approximately constant in time until complete exhaustion, why the migration rates have to be adjusted differentially for these two pesticidal agents and the loading has to be adjusted correspondingly to yield a long term effect with both pesticidal agents. However, in other cases, concentration ratios are desired to vary with time. Parameters that can be varied to achieve the de- sired concentration behaviour with time are, among others, the type of polymer and addition of EVA or PVA and/or the fibre diameter, for example between 0.05 and 0.35 mm.

Additives may be incorporated into the thermoplastic polymer. The following is a non- exhaustive list of possible additives: UV protectors, colorants, fillers, impact modifiers, nucleating agents, coupling agents, conductivity-enhancing agents to prevent static electricity, thermal stabilizers, carbon radical scavengers or oxygen radical scavengers, flame retardants, mould release agents, optical brighteners, antiblocking agents, foam- forming agents, anti-soiling agents, thickeners, further biocides, fragrance.

Due to the satisfactory insecticidal effect of the combination of Abamectin and Fenpyroximate, in some embodiments, the substrate is free or substantially free from other insecticides. Especially, the substrate is free or substantially free from other insecticides in synergistic amounts. The term "substantially free" is meant as a measure, where oth- er insecticides are not present in amounts that are comparable in effect as the combination of the Abemectin and the Fenpyroximate; for example, the substrate may contain trace amounts of other pesticides that do not influence the overall insecticidal efficacy to an extend comparable with the combination of the Abamectin and the Fenpyroximate. For example, the influence of the additional insecticide on the total efficacy may be less than 10% or 1% or 0.1% of the efficacy caused by the specific combination of Abemectin with Fenpyroximate. Abamectin in the context of the invention is following the typical definition in the field, where Abamectin contains more than 80% of Avermectin Bla and less than 20% Avermectin Bib. All percentages are given in weight percentages unless otherwise stated. When weight percentages are given in relation to a thermoplastic polymer, the term "thermoplastic polymer" means the thermoplastic polymer matrix excluding all other ingredients that are not thermoplastic polymer, such as the Abamectin, Fenpyroximate, and optional support particles and other additives.

SHORT DESCRIPTION OF THE DRAWINGS

The invention will be explained in more detail with reference to the drawing, where FIG. 1 illustrates experimental results for Permanet 2.0 in comparison with a fabric containing Abamectin only and a second fabric containing Fenpyroximate as well as Abamectin;

FIG. 2 is illustrating a simulation for the expected long term development of the concentration of Abamectin and Fenpyroximate in a substrate. DETAILED DESCRIPTION / PREFERRED EMBODIMENT

In order to evaluate insecticidal efficacies, a screening of various combinations of Abamectin and Fenpyroximate was performed in order to evaluate the insecticidal efficacy and search for synergistic combinations.

The combination of Abamectin and Fenpyroximate is motivated by the increasing resistance of insects, especially mosquitoes, against currently used insecticides, for example Deltamethrin. As explained above, experiments have revealed that Abamectin yield a high mortality against mosquitoes but slow knock-down efficacy, and Fenpyroximate has a relatively quick knock-down capability but is not an efficient killer.

This is illustrated in FIG. 1, where the knockdown and mortality capabilities against a very pyrethroid-resistant mosquito strain Tiassale are tested; in the test, a net with a deltamethrin (DM) coating was used and two prototypes of fabrics for wall lining, in which one contained Abamectin only and the other contained both Abamectin and Fenpyroximate. The net with the deltamethrin coating was of the type marketed as PermaNet 2.0, being a standard according to WHOPES accreditation. The net is a polyester net with a 36 multifilament 75 Denier yarn, The net is coated with a fluorocarbon coating containing 1.8 g DM per kg. The wall lining prototypes were made from non- woven material, containing spunbond polyethelene fibres of 3 Denier and having a weight of 65 grams per square meter. The Abamectin and Fenpyroximate were incorporated in the polyethylene material with an Abamectin concentration of 2.5 g/kg and a Fenpyroximate concentration of 10 g/kg.

As it appears, the data for the DM net showed some knock down capabilities, but the mortality was low, as the mosquitoes could recover. The Abamectin showed mortality efficacy that increased with time, but which was very slow. The combination of Fenpyroximate and Abamectin yielded a relatively quick knockdown and a final high mortality. Thus, the mosquitoes could not recover from the knockdown but died. This is a very promising result when having fight against malaria in mind.

The combination of Abamectin and Fenpyroximate was tested in a contact bioassay using female Anopheles quadrimaculatus mosquitoes. Other combinations of Abamectin with METI were tested as well and showed results with a different trend. Thus, the behaviour of Abamectin and Fenpyroximate was significantly useful among the various tested combinations.

The insecticides were obtained from commercial sources and dissolved in Dimethyl sulfoxide (DMSO) to generate 10 mg/ml working stocks. Test samples were prepared in a triton/acetone mixture and transferred into a test glass jar that was placed on a test tube rotator or hot dog roller in the fume hood to allow the acetone to evaporate, producing a homogeneous coating of the chemical on the wall and the bottom of the jar. When two insecticides were used in combination, they were mixed at the appropriate fixed ratios, and the jar was coated as described above. Mosquitoes (3-5 day old) were collected in a vial, chilled on ice to immobilize them, and 10 females were introduced into the compound treated jar through the slit on the covering mesh. A moist cotton ball was used to cover the slit and knockdown/mortality assessed at 1, 6, and 24 hrs post exposure. Below, efficacy results are presented for insecticidal exposure of the mosquito strain Anopheles quadrimaculatus.

Dose response was conducted to obtain EC50 concentration values for Abamectin as well as for Fenpyroximate and, subsequently, for the combination in order to evaluate possible synergistic effects. EC50 is the "Effective Concentration" for killing 50% of the exposed insects. Correspondingly EC75 and EC90 concentration values were obtained as well for the agents. The experimental individual approximate EC50, EC75, and EC90 are given in the table below.

From the measured EC50 concentrations for the individual agents as well as for the combinations, Combination Index (CI) values were determined following the principles outlined in the article "An overview of Drug Combination Analysis with Isobolograms" by R.J.Tallida, published in The Journal of Pharmacology and Experimental Therapeutics, Vol. 319, No. 1, 2006, pagesl-7.

To obtain the CI values for a specific combination, a start concentration for each agent was used that was higher than the individual EC50 value for the substance. In one of experiments for the combination of Abamectin and Fenpyroximate, a start concentration of 5 ppm was selected for Abamectin, which is higher than the EC50 value of 3.7 ppm, and a start concentration of 50 ppm was selected for Fenpyroximate, which is higher than its EC50 value of 21 ppm; with these initial concentrations, daily specific EC50 values were estimated. These values of 5 ppm and 50 ppm for Abamectin and Fenpyroximate, respectively, were used as a starting point for a mixture of 5 ppm Abamectin and 50 ppm Fenpyroximate and used for a series of experiments, where the mixture was diluted 6 times by a dilution factor of 2 in order to find the EC50 concen- tration for the combination. This EC50 value was then compared to the EC50 values for the case where the two insecticides are not combined. Likewise for the EC75 and EC90 values, where the concentration is higher. From this comparison, a Comparative Index CI could be determined. A CI value of around 1 suggests that compounds function additively. Synergistic combinations result in a CI<1 and antagonistic behavior is deduced for compounds with a CI>1. Due to the statistical uncertainty of data when dealing with biological systems, data up to 2 are regarded as still within the additive regime, whereas CI values above 2 are regarded as antagonistic. These definitions determine the concentration ratios that are regarded as being in the additive regime or in the antagonistic regime.

The experimental series were conducted for start values of 7.5 ppm Abamectin and 25 ppm Fenpyroximate, 5 ppm Abamectin and 50 ppm Fenpyroximate, and 2.5 ppm Abamectin and 75 ppm Fenpyroximate. Synergistic behavior is marked by numbers in italics.

As it appears the combination of Abamectin and Fenpyroximate has an additive effect if the ratio of Abamectin to Fenpyroximate is less than approximately 1 :3, and a synergistic effect on mosquitoes if the ratio of Abamectin to Fenpyroximate is less than 1 :10.

Once, the synergistic effect has been observed by the above method, the necessary ratios for synergy between the Abamectin and Fenpyroximate can be calculated in addition to the concentrations that are necessary against the specific insect. Performing similar experiments for other mosquito strains extends the picture for the necessary concentrations and ratios. The values from the above table are ratios that relate to exposure values in the specific experiment. Whereas the ratios are generally valid for various concentrations, the concentrations as such in the formulations that were used in the experiments would have to be re-defined in the insecticidal product, for example a mosquito net. It would also have to be re-defined in dependence on whether the substrate, for example a mosquito net, would be coated, or the insecticides incorporated in the bulk material of the fibres. Thus, the concentration, as found in the experiments, translates not directly to the necessary concentration in a product. However, the ratios are generally applicable over a wide range of concentrations, such that the ratios from the experiments also can be used for determining beneficial ratios in the final product, for example the concentration ratios of the melt-incorporated pesticidal agents.

With offset in the synergy values as deducted above, bulk concentrations have been determined and tested in order to find appropriate ranges for bulk concentrations. On the one hand, the bulk concentrations have to be high enough to yield long term efficacy, on the other hand, the concentration should not be unnecessarily high in the bulk, as this increases production costs and may violate international restriction on allowable maximum values for load levels in insecticidal bulk materials, such as mosquito nets. Such ranges for ratios and concentrations in bulk material are given for textiles, where fibres are extruded with insecticide incorporated in the fibres. Such fibres are then used for mosquito nets and wall linings, for example. The ratios apply also for tarpaulins and foils used in dwellings, although there may be used a slight adjustment according to the specific product. For outdoor use, where UV degradation influences the breakdown of the insecticide, the bulk concentrations may be accordingly higher in order to have a sufficiently large buffer for long-lasting replenishing on the surface of the substrate.

Due to additive effects, relatively high concentrations of the agents often lead to death of the mosquitoes, despite being outside the range of ratios for synergistic effects. The synergistic effect is important when the exposure dose becomes low. Often, this is exactly the objective, namely, to enhance the efficacy at low concentrations. These considerations have been used to develop a specific long term model that is explained with reference to the drawing of FIG. 2. The drawing shows a simulation for the decrease of Abamectin (Aba) and Fenpyroximate (FNP) released from a substrate that is a needle punch nonwoven prototype in which the two substances are incorpo- rated. As it is seen, the release of Fenpyroximate is slower over the years than the release of Abamectin. This means that the ratio between the surface concentration of Abamectin and Fenpyroximate is gradually reduced. While being reduced, the ratio comes from an initial ratio of 1 :4 into the synergistic regime, where the ratio is less than 1 : 10. Thus, the reduction of lethal efficacy by Abamectin is substituted by a syn- ergistic lethal effect caused by the combination.

This model implies the following. In the initial state of the fabric, the concentration in the bulk and, correspondingly, on the surface is high, and a good lethal effect on the insect is obtained either by one of the insecticidal agents or by an additive effect. Once, the concentrations decrease over the years, the lower concentration is balanced by a higher relative efficacy due to the synergistic effect that takes offset at lower concentration, as also shown in the experiments described above.

The model solved the problem of early decrease of efficacy due to concentrations that fall under a lethal level. However, in order to obtain that model, it must be assured that the long term release of Fenpyroximate is slower than the release of Abamectin. For example, this can be promoted by loading Fenpyroximate onto a support particle, for example clay which acts as a slow-release buffer of the clay. The net effect is a gradual development of the insecticidal system into the synergistic region. In the specific model as illustrated in the drawing, the ratio crosses the 0.1 ordinate after 2.5 years, such that a synergistic effect can be expected to substantially influence the efficacy after about 3 years.

As the concentration of the Fenpyroximate is maintained at a relatively high level, the knock-down efficacy is maintained, whereas the lethal effect undergoes the change from the additive to a synergistic effect. As a conclusion, the considerations for the insecticidal efficacy for the combination of Abamectin and Fenpyroximate against mosquitoes are complex and take into regard maintaining a high knock-down level and a high lethal effect on a long term perspective despite gradual exhaustion.

Claims

1. A method for killing adult mosquitoes, the method comprising providing a nonliving substrate having Abamectin and Fenpyroximate on a surface of the substrate; exposing the adult mosquito to a Abamectin and Fenpyroximate in combination by contact of the adult mosquito with the surface.

2. A method according to claim 1, wherein the method comprises providing the Abamectin and the Fenpyroximate on the surface at a surface concentration ratio between the surface concentration of Abamectin and the surface concentration of Fenpyroximate, wherein the surface concentration ratio exhibits additive or synergistic effects on the mosquito and not antagonistic effects.

3. A method according to claim 2, wherein the surface concentration ratio is in the range of 1 : 1000 to 1 :3.

4. A method according to claim 2, or 3, wherein the method comprises selecting a spe- cific strain of mosquitoes, determining a concentration ratio for the specific strain which exhibits additive or synergistic killing effect on the mosquito strain, and adjusting the surface concentration ratio for additive and synergistic killing effect.

5. A method according to anyone of the claims 2 to 5, wherein the method comprises providing the substrate with a surface concentration ratio that causes an additive killing effect on the mosquito and causing a gradual change of the surface concentration ratio into a synergistic effect.

6. A method according to any preceding claim, wherein method comprises providing the substrate as a thermoplastic polymer substrate.

7. A method according to claim 6, wherein the method comprising melt-incorporating Abamectin and Fenpyroximate in the thermoplastic polymer of the substrate, extruding or moulding the thermoplastic polymer with the incorporated Abamectin and Fenpyroximate as part of the production of the substrate; and causing Abamectin and Fenpy- roximate to migrate to the surface of the substrate.

8. A method according to claim 7 wherein the method comprises providing a bulk concentration ratio R between the bulk concentration of Abamectin and the bulk concentration of Fenpyroximate, where R is in the range of 1 : 1000 to 1 :3.

9. A method according to claim 8, wherein the method comprises providing the thermoplastic polymer of the substrate with a concentration of Abamectin in the interval of 0.25-25g per kg thermoplastic polymer of the substrate and a concentration of Fenpyroximate in the interval of 1-50 g per kg of thermoplastic polymer of the substrate.

10. A method according to claim 9, wherein the method comprises loading Fenpyroximate on support particles and incorporating the loaded support particles into the thermoplastic polymer.

11. A method according to claim 10, wherein the method comprises dividing an amount of Fenpyroximate in to a first portion and a second portion, the first portion being 20%-80% of the amount of Fenpyroximate, loading only the first portion on support particles and melt-incorporating the support particles loaded with the first portion as well as the second portion of Fenpyroximate.

12. A method according to any one of the claims 6-9, wherein the method comprises incorporating Abamectin but not Fenpyroximate in a first type of extruded fibres and Fenpyroximate but not Abamectin in a second type of extruded fibres, and combining the two types of fibres into a textile as the substrate or as part of the substrate.

13. A method according to claim 12, wherein the method comprises loading Fenpyroximate on support particles and incorporating the loaded support particles into the thermoplastic polymer of the second type of fibres.

14. A method according to claim 12 or 13, wherein the method comprises dividing an amount of Fenpyroximate in to a first portion and a second portion, the first portion being 20%-80% of the amount of Fenpyroximate, loading only the first portion on support particles and incorporating the support particles loaded with the first portion as well as the second portion of Fenpyroximate into the molten thermoplastic polymer for the second type of fibres; and then extruding of the second type of fibres.

15. A substrate comprising a thermoplastic polymer having incorporated therein Abamectin and Fenpyroximate for migration from a bulk of the substrate to a surface of the substrate.

16. A substrate according to claim 14, wherein the range of ratios R between the bulk concentration of Abamectin and the bulk concentration of Fenpyroximate is in the range of 1 : 1000 to 1 :3

17. A substrate according to claim 14 or 15, wherein the bulk concentration of Abamectin is in the range 0.25-25, and the bulk concentration of Fenpyroximate is in the range of 1-50 in terms of g/kg of the bulk polymer of the substrate excluding the weight of Abamectin and Fenpyroximate.

18. A substrate according to any one of the claims 14-16, wherein the substrate is a textile comprising a first type of thermoplastic fibres containing Abamectin but not Fenpyroximate in the thermoplastic bulk of the first type of fibres and comprising a second type of thermoplastic fibres containing Fenpyroximate but not Abamectin in the thermoplastic bulk of the second type of fibres.

19. A substrate of claim 17, wherein the second type fibres comprises support particles loaded with Fenpyroximate, wherein the support particles are incorporated into the bulk of the second type of thermoplastic fibres.

20. A substrate according to any one of the claims 14-18, wherein the substrate is a textile, the textile being a mosquito net or a wall lining.

21. Use of a combination of Abamectin and Fenpyroximate for killing mosquitoes.