Method for determining penetration properties of a compound

Abstract

A method for determining the penetration properties of a compound into and / or through a plant leaf wax cuticular layer, said method comprising a) providing a model plant wax layer comprising at least one wax and Nile Red; b) subsequently contacting the model plant wax layer with a composition comprising the compound; and c) monitoring the penetration of the compound into and / or through the model plant wax layer by monitoring the change in Nile Red fluorescence. Furthermore, the use of Nile Red comprised in a wax layer for determining the penetration properties of a compound into and / or through said wax layer is described, and a wax layer deposited onto a solid support, wherein said wax layer comprises at least one plant wax and Nile Red.

Description

[0001] METHOD FOR DETERMINING PENETRATION PROPERTIES OF A COMPOUND

[0002] TECHNICAL FIELD

[0003] The present invention lies in the field of agrochemical sciences and relates to a method for determining the permeation and penetration properties of a compound into and through cuticular wax layers. The methods described herein comprise the steps of: a) providing a model plant wax layer comprising at least one wax and Nile Red (9-(Diethylamino)-5 / 7-benzo[a]phenoxazin-5-one); b) contacting the model plant wax layer with a composition comprising the compound; and c) monitoring the penetration of the compound into and / or through the model plant wax layer by monitoring the Nile Red fluorescence. Also encompassed are the related uses and the wax layer as such.

[0004] BACKGROUND OF THE INVENTION

[0005] Leaves of all land plants are covered by an extracellular hydrophobic layer, plant cuticle, protecting them against desiccation and external environmental stresses. Control over the permeability through leaf cuticles is of great importance in an effort to increase the sustainability of agriculture and feeding of the world’s population.

[0006] Currently, this control is ensured by the application of complex formulations where adjuvants play a crucial role in increasing the permeability of the active ingredient through the leaf cuticle, which is the transportlimiting barrier for systemic pesticides. Additionally, these formulations improve wetting and adhesion of sprayed droplets on leaf surfaces.

[0007] Efforts have been taken to better understand the structure and dynamic properties of epicuticular wax, the outer surface of the leaf cuticle, which is a prerequisite for the development of novel adjuvants with the desired properties and effective softening of plant leaves’ cuticles. While the understanding of the epicuticular wax layer and its properties has improved over recent years, there is still need in the art for methods that allow assessing and monitoring the capability of a compound that is either known or suspected of having useful adjuvant properties to penetrate into and through the cuticular wax layer.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention meets this need by providing a method that allows determining the penetration / permeation properties of a compound into and / or through the leave’s cuticular wax layer. To achieve this, the inventors of the present invention have developed a model of a plant wax layer that allows monitoring the penetration of an adjuvant or any other compound into the wax layer via Nile Red fluorescence in the wax layer. This model is based on the surprising finding that compounds that penetrate into a wax layer stained with Nile Red have measurable influence on the nanoenvironment of the dye in that the quenching properties of the wax are reduced and a detectable increase in fluorescence in the direct vicinity is observed. This method allows screening the penetration properties of different compounds and compositions, for example even in a high-throughput approach, and thus facilitates the development and selection of adjuvants that promote the penetration of active ingredients.

[0010] In a first aspect, the present invention therefore relates to a method for determining the penetration properties of at least one compound into and / or through a plant leaf wax cuticular layer, said method comprising a) providing a model plant wax layer comprising at least one wax and Nile Red (9-(Diethylamino)-5 / 7- benzo[a]phenoxazin-5-one), optionally on a solid support, wherein the Nile Red is optionally homogeneously distributed in the wax layer; b) subsequently contacting the model plant wax layer with a composition comprising the at least one compound; and c) monitoring the penetration of the at least one compound into and / or through the model plant wax layer by monitoring the Nile Red fluorescence, preferably the change in Nile Red fluorescence caused by the at least one compound.

[0011] In various embodiments of the method, the compound is a potential adjuvant for agrochemical formulations, preferably a penetration enhancing adjuvant.

[0012] It is preferred that the at least one wax of the model plant wax layer is a plant wax, preferably a plant wax having a melting point in the range of 40 to 90 °C and / or a hydrocarbon length of 26 to 35 carbon atoms. The plant wax may be carnauba wax, candelilla wax, or a combination of waxes comprising carnauba wax, candelilla wax or both.

[0013] In various embodiments, the model plant wax layer has a thickness of 1 to 30 pm, preferably 2 to 20 pm, more preferably 3 to <20 pm.

[0014] In various embodiments, the model plant wax layer comprises, relative to its total weight, 1 to 1000 ppm, preferably 50 to 750 ppm, more preferably 200 to 600 ppm, Nile Red.

[0015] The model plant wax film may be provided on a solid support. Said solid support may be glass, preferably a glass microscope slide.

[0016] In various embodiments of the method, the contacting occurs with the surface of the model plant wax layer opposite to the surface contacting the solid support.

[0017] It is preferred that the composition comprising the at least one compound is a liquid composition, optionally an aqueous solution or dispersion comprising said at least one compound.

[0018] In various embodiments, the composition comprises the at least one compound, which may be a penetration enhancing adjuvant for agrochemical formulations, and an agrochemical active. In various embodiments, contacting is carried out for up to 24 hours, for example 10 minutes to 22 hours.

[0019] The penetration of the compound into and / or across the model plant wax layer can be monitored using fluorescence microscopy, preferably confocal microscopy.

[0020] Nile Red fluorescence can be determined, in various embodiments, at an excitation wavelength of about 561 nm and / or an emission wavelength of about 565 to about 750 nm.

[0021] In various embodiments, the penetration properties of said compound are determined by

[0022] (1) measuring the change in Nile Red fluorescence intensity,

[0023] (2) measuring and calculating the Nile Red fluorescence intensity ratio between a first channel in the range of about 565 to about 645 nm and a second channel in the range of about 650 to about 745 nm, and / or

[0024] (3) Nile Red fluorescence lifetime imaging (FLIM).

[0025] Preferably, the method is a screening method to determine the penetration properties of a multitude of compounds, preferably in a multiplexing format.

[0026] In another aspect, the present invention relates to the use of Nile Red comprised in a wax layer, optionally homogeneously distributed therein, for determining the penetration properties of at least one compound into and / or through said wax layer, optionally by monitoring the change in Nile Red fluorescence upon contacting the at least one compound with the wax layer.

[0027] In still another aspect of the present invention, it relates to a wax layer, optionally deposited onto a solid support, wherein said wax layer comprises at least one plant wax and Nile Red, wherein the Nile Red is optionally homogeneously distributed in the wax layer, and wherein said solid support is preferably a glass slide. In various embodiments, the solid support is not plant material, such as a plant leaf.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows a schematic illustration of the principle of confocal laser scanning microscopy (CLSM) for the excitation path (left hand side) and detection path (right hand side) for an inverted microscope setup.

[0030] Figure 2 (schematically) shows the structure of a plant leaf.

[0031] Figure 3 shows fluorescence intensity and FLIM imaging as described in the Examples. Scale bars are 20 pm.

[0032] Figure 4 shows a schematic illustration of the penetration of an adjuvant into a Nile Red (stars) stained wax layer with crystalline portions (bars) (Figure 4A) as well as a confocal microscopy image of a cross section of a Nile Red stained wax layer. Figure 5 shows the Nile Red fluorescence spectrum and the observed redshift upon polarity increase. While the drawing shows the separation between short channel and long channel wavelength range to be at about 600 to 605 nm, the preferred threshold between short and long channel is at about 645 nm, as described in the examples.

[0033] Figure 6 shows the ratiometric analysis results as fluorescence ratios over time at different depth after contacting 1000 ppm Plurafac® LF431 in water with the wax layer.

[0034] Figure 7 shows penetration of the active model ingredient NBD-Pz into the wax layer after an exposure time of 1 h with Plurafac® LF 1300 (adjuvant). 8A shows Nile Red at 561 nm; 8B shows NBD-Pz at 488 nm and 8C NBD-Pz at 488 nm with maximum projection.

[0035] Figure 8 shows penetration of Coumarin 343 into the wax layer after an exposure time of 1 h with Plurafac® LF 1300 (adjuvant). 9A shows Nile Red at 561 nm; 9B shows Coumarin 343 at 405 nm and 9C Coumarin 343 at 488 nm with maximum projection.

[0036] Figure 9 shows penetration of the non-polar model active ingredient Fluorol Yellow into the wax layer after an exposure time of 1 h with Plurafac® LF 1300 (adjuvant). 10A shows Nile Red at 561 nm; 10B shows Fluorol Yellow at 488 nm and 10C Fluorol Yellow at 488 nm with maximum projection.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] All technical and scientific terms used herein have, unless explicitly stated otherwise, the meanings as commonly understood in the art.

[0039] The singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.

[0040] “At least one” as used herein, means one or more, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more of the referenced species. Similarly, “one or more”, as used herein, relates to at least one and comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more. In connection with a given species, the term does not relate to the total number of molecules, but rather to the type of species. “At least one compound”, for example, thus means that one type of compound or two or more different types of compounds may be present. In connection with amounts, the term relates to the total amount of the referenced species.

[0041] Numeric values specified without decimal places here refer to the full value specified with one decimal place, i.e., for example, 99 % means 99.0 %, unless otherwise defined.

[0042] All percentages given herein in relation to compositions or formulations relate to weight % (wt.-%) relative to the total weight of the respective composition or formula, if not explicitly stated otherwise. Numeric ranges specified in the format “from x to y” include the specified values. If multiple preferred numeric ranges are specified in this format, it is understood that all ranges created by combining the different endpoints are also included.

[0043] The terms “about”, “approximately” or “approx.”, in connection with a numerical value, refer to a variance of ± 10 %, preferably ± 5 %, with respect to the given numerical value. In case two adjacent ranges of values are given, the term may be interpreted such that no overlap occurs.

[0044] It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

[0045] Furthermore, the terms "first", "second", "third" or "a.", "b.", "c.", "d.", “(1)”, “(2)”, etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms "first", "second", "third" or "a.", "b.", "c.", "d.", “(1)”, “(2)”, etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.

[0046] It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention that will be limited only by the appended claims.

[0047] In a first aspect, the present invention relates to a method for determining the penetration properties of at least one compound into and / or through a plant leaf wax cuticular layer, said method comprising a) providing a model plant wax layer comprising at least one wax and Nile Red (9-(Diethylamino)-5 / 7- benzo[a]phenoxazin-5-one), optionally on a solid support, wherein the Nile Red is optionally homogeneously distributed in the model plant wax layer; b) subsequently contacting the model plant wax layer with a composition comprising the compound; and c) monitoring the penetration of the compound into and / or through the model plant wax layer by monitoring the change in Nile Red fluorescence caused by the at least one compound.

[0048] The method aims to assess the penetration or permeation properties of a compound or a combination of compounds, which may be part of a composition, into a model wax layer that is designed to resemble a plant cuticular wax layer. “Into” the layer, as used herein, means permeation from outside the layer into the material of the layer. “Through” or “across” as used in relation to penetration / permeation means passing completely through the layer to end up at the other side of the layer. If the layer is deposited on a solid support, said term “across” may also mean that the respective compound(s) accumulate at the boundary between the wax layer and the support layer. The terms “penetration” and “permeation” as used interchangeably herein, relate to the property of migrating into and optionally through a material, i.e. the wax layer. As this term is used in relation to chemical compounds, it means that the chemical compound moves, for example by diffusion, from outside a material into the material such that it ends up in between the molecules that form the material. Said movement is typically not driven by any chemical or physical interaction between the penetrating molecule and the material but rather by diffusion or similar force.

[0049] “Homogenously distributed”, as used herein in connection with the Nile Red, means that the Nile Red is distributed within the wax layer such that its concentration is essentially identical at each location within the wax layer. “Essentially identical” means that there may a variation of no more than ±20%. The homogenous distribution ensures that there is a homogenous base fluorescence across the entire wax layer, which in turn allows to trace the penetration of any compound that (locally) affects the base fluorescence provided by the Nile Red. However, as the fluorescence is monitored prior to and after / during contacting with the composition / compound of interest, an inhomogeneous distribution may also suffice as the interaction with the compound would still cause a local change in fluorescence of the Nile Red. In case the wax layer has crystalline, semicrystalline and / or amorphous parts, it has been found that the Nile Red is particularly located in the amorphous parts, where it is typically distributed homogeneously. In various embodiments, the Nile red is thus preferably homogeneously distributed in the amorphous phases of the wax layer.

[0050] The readout of the methods of the invention is the change in Nile Red fluorescence caused by the proximity of a compound of interest. In other words, undisturbed Nile Red provides for a base fluorescence level that is essentially the same at any random location within the wax layer (due to its homogenous distribution therein). Once a compound contacts and penetrates into the wax layer, it interacts with the Nile Red and locally changes the fluorescence signal. Its penetration into and optionally across the wax layer may thus be monitored / traced by monitoring the local change in Nile Red fluorescence.

[0051] The at least one compound of which the penetration properties are determined may be any chemical compound or may comprise multiple compounds, such as for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different compounds. The compound may be a component of a composition that comprises other compounds. In order to determine the penetration properties of a specific compound or a combination of specific compounds typically a comparison to the same composition without these compounds is made to allow determining the penetration properties of the at least one compound or its contribution to the penetrating properties of the composition.

[0052] As the method particularly aims to allow identifying or screening for compounds that increase the permeability of the leaf cuticle, in particular the permeability not only for itself but also for an active ingredient, such as an active agrochemical ingredient, for example a pesticide, the at least one compound may be a known adjuvant in agrochemical compositions or a compound suspected of being useful as an adjuvant in agrochemical compositions, i.e. a potential adjuvant for agrochemical compositions. The at least one compound is preferably a compound that has or is suspected to have penetration enhancing properties, i.e. increases permeability of the leaf cuticle, as determined by means of the model wax layer. Such compounds that are confirmed to have the capability of increasing permeability of the leaf cuticle - at least in the model system - are also referred to as “penetration enhancing adjuvants”.

[0053] The at least one compound tested or screened may be any type of compound. In various embodiments, it is an organic compound. In various embodiments, it may have emulsifying or surfactant properties. In various embodiments, it may have a hydrophobic moiety as part of the molecule that facilitates penetration into a hydrophobic wax layer. In various embodiments, it may optionally also have hydrophilic properties to allow dissolution or dispersing in an aqueous phase. In various embodiments, the compound is uncharged, i.e. nonionic, at pH 7 or at least in a pH range of 5 to 9 or 4 to 10 or 3 to 11 . Suitable types of molecules include, without limitation, alkoxylated fatty acids or fatty alcohols or oxo alcohols, which may be branched or unbranched, carboxylic acid (such as C8-C20 carboxylic acids) esters or amides, ethoxylated or epoxidized oils, alkyl(poly)glycosides and the like. “Alkoxylation” may be ethoxylation, propoxylation, butoxylation or any combination thereof. Suitable types of compounds are the Plurafac® line of molecules, as obtainable from BASF SE, in particular the Plurafac® LF line of products, but also compounds of the Agnique® and Lutensol® product lines. In various embodiments, the compound tested is itself not an agrochemical active, i.e. is preferably different from the types and examples of agrochemical actives disclosed herein below.

[0054] The model plant wax layer is designed to mimic and resemble the properties of a plant leaf’s cuticle and comprises at least one wax, which may be a synthetic or plant wax or a mixture thereof. In various embodiments, it essentially consist of said at least one wax. “Essentially consists of’, as used herein, relates to amounts of more than 50 wt.-%, typically at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 wt.-% relative to the total weight of the reference composition / material. Accordingly, it is preferred that the model wax layer comprises the at least one wax in an amount of more than 50, typically more than 70, preferably more than 80, more preferably more than 85 % by weight. It may comprise further components aside the at least one wax and the Nile Red.

[0055] In various embodiments, it is preferred that the at least one wax of the model plant wax layer is a plant wax. The plant wax can be any suitable plant wax or mixture of plant waxes. In various embodiments, the plant wax has a melting point in the range of 40 to 90 °C, for example from 45 to 90 °C, 50 to 90 °C, 55 to 90 °C, 60 to 90 °C, or 65 to 90 °C, preferably from 67 to 88 °C, more preferably from 70 to 87 °C, most preferably from 75 to 87 °C such as 80 to 87 °C or 82 to 86 °C, and / or a hydrocarbon length of 26 to 35 carbon atoms, preferably from 27 to 34 carbon atoms such as 28 to 34 carbon atoms. In preferred embodiments, the plant wax is carnauba wax from Copernicia prunifera. Such wax is well known and widely used in the art (for different purposes) and is commercially obtainable, e.g., from Alfa Aesar. It has been found that carnauba wax is particularly suitable as a model for a plant leaf’s cuticular layer, as it closely resembles its typical characteristics or at least closely resembles the properties of the cuticular layer of typical important crop plants, such as wheat or corn or soybean. In other embodiments, the plant wax is candelilla wax from Euphorbia antisyphilitica. Such wax is also well-known and used as a food additive or in the cosmetic industry. In various embodiments, combinations of carnauba wax and candelilla wax max be used either alone or in combination with further waxes.

[0056] The used wax or wax mixture preferably has a high ester content of at least 70 % or higher, preferably at least 75 % or higher and most preferably at least 80 % or higher, e.g., at least 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 % 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 % 96 %, 97 %, 98 %, or 99 %. The used wax or wax mixture may also have a high crystallinity, for example of at least 60%, at least 65%, at least 70% or at least 75%.

[0057] In various embodiments, the model plant wax layer is provided in a thickness that is similar to that of a plant leaf’s wax layer. Therefore, in various embodiments, it is provided in form of a layer with a thickness of 1 to 30 pm, preferably 2 to 20 pm, more preferably 3 to <20 pm. The upper limit may be 19, 18, 17, 16, 15, 14, 13 or 12 pm and the lower limit may be 3, 4, 5, 6, 7, or 8 pm. In various embodiments, its thickness is about 10 pm ± 5pm. The wax layer may be a wax film and the terms “layer” and “film” are used interchangeably herein.

[0058] As described above, the wax layer may comprise additional components, such as, for example, small amounts of water. In various embodiments, the wax layer consists essentially of the at least one plant wax and Nile Red, for example to an extent that at least 80, preferably at least 90 wt.-%, more preferably at least 95 wt.-% relative to the total weight of the wax layer are the at least one plant wax and the Nile Red.

[0059] The wax layer comprises 1 to 1000 ppm Nile Red relative to the total weight of the wax layer.

[0060] Nile Red is chemically 9-(Diethylamino)-5 / 7-benzo[a]phenoxazin-5-one and sometimes abbreviated as “NR”. It is a hydrophobic, highly fluorescent, solvatochromic dye of Formula (I)

[0061] Its content in the wax layer is selected such that the penetration properties of the tested compound can be determined by means of monitoring NR fluorescence, which changes upon penetration of another compound into the wax layer.

[0062] In various embodiments, the amount of Nile Red in the wax layer, relative to its total weight, is in the range of 50 to 900 ppm, 50 to 800 ppm, 50 to 750 ppm, 75 to 700 ppm, 75 to 700 ppm, 100 to 650 ppm, 100 to 600 ppm, 150 to 600 ppm or 200 to 600 ppm. The lower limit may be 50, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 ppm, and the upper limit may be 1000 ppm, 950 ppm, 900 ppm, 850 ppm, 800 ppm, 750 ppm, 700, or 650 ppm. Typically, ranges of 200 to 600 or 300 to 700 ppm or levels of about 500 ppm are used. In various embodiments, the model plant wax layer is prepared by melting the wax, optionally adding further components, such as the Nile Red to the melt, and depositing the melt onto a solid support, where it may cool and harden. The depositing may be done by pouring, dipping, spraying or any other known and suitable method. Typically, the obtained wax film is doctor bladed to adjust its thickness and uniformity. In various embodiments, the obtained layer can be further processed by thermal curing techniques. By adding the Nile Red to the melt, its homogenous distribution therein may be achieved.

[0063] The model plant wax film may be provided on a solid support. The solid support may be any suitable material but is preferably glass or another material that is suitable for use in fluorescence microscopy. As the evaluation and monitoring of the fluorescence is typically made via confocal microscopy, the solid material needs to be suitable for these methods. In various embodiments, the solid support is a microscope slide, such as a glass microscope slide. In various embodiments, the solid support is not plant material, in particular no natural plant material, such as a plant leaf or part thereof.

[0064] In step b) of the described methods, the wax layer in which the Nile Red is optionally homogeneously distributed, is then contacted with the at least one compound, which is typically comprised in a composition. In some embodiments, the composition may consist of the at least one compounds, but typically it comprises the at least one compound and a solvent or carrier medium. Said solvent or carrier medium may be a liquid, for example an aqueous solvent or medium. “Liquid” as used in this context, relates to compositions that are liquid under standard conditions, i.e. at 20°C and 1013 mbar. The aqueous solvent may be water or a mixture of water and an organic solvent, such as an alcohol. In other embodiments, the solvent may be an organic solvent. However, the solvent or carrier medium needs to be compatible with the wax layer in that it does not dissolve or disperse or in other ways adversely affects the integrity of the wax layer, also some swelling may be acceptable.

[0065] The at least one compound to be tested for its penetration properties can be used in any suitable concentration. Generally, typically used concentration ranges are between 10 and 100.000 ppm, for example between 100 and 10000 ppm. In various embodiments, concentrations of the at least one compound in the composition of about 500 to about 5000 ppm, relative to the total weight of the composition, are used.

[0066] The composition may, in addition to the at least one compound to be tested, and the optional carrier or solvent comprise additional components, in particular if the compound is part of an agrochemical formulation. Typical ingredients of such formulations are generally known in the art and include, among others, compounds selected from the group consisting of co-solvents, dispersants, further adjuvants, surfactants, thickeners, humectants, antioxidants, colorants, fragrances, defoamers, anti-caking agents, biocides, anti-freeze agents, emulsifiers, and mixtures thereof.

[0067] Co-solvents may be selected from fatty acid methyl esters, aromatic hydrocarbons, 2-ethylhexanol esters, dimethyl fatty amides, dimethyl alkyl amides, fatty alcohols, glycols, alkyl esters, lactate esters, aromatic esters, alkyl pyrrolidones, paraffinic mineral oils, vegetable oils, lactones, carbonates, carbamates different from the carbamate-based solvents of formula (I), alkyl amides, ketones, phosphate esters, aldehydes, ethers, acetals, and mixtures thereof.

[0068] Suitable emulsifiers and / or surfactants include non-ionic and anionic surfactants and their mixtures. The following list of compounds include compounds that can be used as further ingredients of the composition to be tested but may also be the at least one compound tested for its penetration properties.

[0069] Non-ionic surfactants include for example:

[0070] - products of the addition of 2 to 30 mol ethylene oxide and / or 0 to 5 mol propylene oxide onto linear Ca-22 fatty alcohols, onto C12-22 fatty acids and onto alkyl phenols containing 8 to 15 carbon atoms in the alkyl group;

[0071] - C12 / 18 fatty acid monoesters and diesters of addition products of 1 to 30 mol ethylene oxide onto glycerol;

[0072] - glycerol mono- and diesters and sorbitan mono- and diesters of saturated and unsaturated fatty acids containing 6 to 22 carbon atoms and ethylene oxide addition products thereof;

[0073] - addition products of 15 to 60 mol ethylene oxide onto castor oil and / or hydrogenated castor oil;

[0074] - polyol esters and, in particular, polyglycerol esters such as, for example, polyglycerol polyricinoleate, polyglycerol poly-12-hydroxystearate or polyglycerol dimerate isostearate. Mixtures of compounds from several of these classes are also suitable;

[0075] - addition products of 2 to 15 mol ethylene oxide onto castor oil and / or hydrogenated castor oil;

[0076] - partial esters based on linear, branched, unsaturated or saturated C6 / 22 fatty acids, ricinoleic acid and 12- hydroxystearic acid and glycerol, polyglycerol, pentaerythritol, dipentaerythritol, sugar alcohols (for example sorbitol), alkyl glucosides (for example methyl glucoside, butyl glucoside, lauryl glucoside) and polyglucosides (for example cellulose);

[0077] - mono-, di- and trialkyl phosphates and mono-, di- and / or tri-PEG-alkyl phosphates and salts thereof;

[0078] - wool wax alcohols;

[0079] - polysiloxane / polyalkyl polyether copolymers and corresponding derivatives;

[0080] - mixed esters of pentaerythritol, fatty acids, citric acid and fatty alcohol and / or mixed esters of C6-22 fatty acids, methyl glucose and polyols, preferably glycerol or polyglycerol;

[0081] - polyalkylene glycols.

[0082] The addition products of ethylene oxide and / or propylene oxide onto fatty alcohols, fatty acids, alkylphenols, glycerol mono- and diesters and sorbitan mono- and diesters of fatty acids or onto castor oil are known commercially available products. They are homologue mixtures of which the average degree of alkoxylation corresponds to the ratio between the quantities of ethylene oxide and / or propylene oxide and substrate with which the addition reaction is carried out.

[0083] Preferred emulsifiers are described in more detail as follows:

[0084] In various embodiments, the emulsifier or surfactant can be a partial glyceride. Typical examples of suitable partial glycerides are hydroxystearic acid monoglyceride, hydroxystearic acid diglyceride, isostearic acid monoglyceride, isostearic acid diglyceride, oleic acid monoglyceride, oleic acid diglyceride, ricinoleic acid monoglyceride, ricinoleic acid diglyceride, linoleic acid monoglyceride, linoleic acid diglyceride, linolenic acid monoglyceride, linolenic acid diglyceride, erucic acid monoglyceride, erucic acid diglyceride, tartaric acid monoglyceride, tartaric acid diglyceride, citric acid monoglyceride, citric acid diglyceride, malic acid monoglyceride, malic acid diglyceride and technical mixtures thereof which may still contain small quantities of triglyceride from the production process. Addition products of 1 to 30, and preferably 5 to 10, mol ethylene oxide onto the partial glycerides mentioned are also suitable.

[0085] In various embodiments, the emulsifier or surfactant can be a sorbitan ester. Suitable sorbitan esters are sorbitan monoisostearate, sorbitan sesquiisostearate, sorbitan diisostearate, sorbitan triisostearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan dioleate, sorbitan trioleate, sorbitan monoerucate, sorbitan sesquierucate, sorbitan dierucate, sorbitan trierucate, sorbitan monoricinoleate, sorbitan sesquiricinoleate, sorbitan diricinoleate, sorbitan triricinoleate, sorbitan monohydroxystearate, sorbitan sesquihydroxystearate, sorbitan dihydroxystearate, sorbitan trihydroxystearate, sorbitan monotartrate, sorbitan sesquitartrate, sorbitan ditartrate, sorbitan tritartrate, sorbitan monocitrate, sorbitan sesquicitrate, sorbitan dicitrate, sorbitan tricitrate, sorbitan monomaleate, sorbitan sesquimaleate, sorbitan dimaleate, sorbitan trimaleate and technical mixtures thereof. Addition products of 1 to 30, and preferably 5 to 10, mol ethylene oxide onto the sorbitan esters mentioned are also suitable.

[0086] In various embodiments, the emulsifier or surfactant can be an alk(en)yl oligoglycoside. The alkyl or alkenyl oligoglycosides representing also preferred emulsifiers may be derived from aldoses or ketoses containing 5 or 6 carbon atoms, preferably glucose. Accordingly, the preferred alkyl and / or alkenyl oligoglycosides are alkyl or alkenyl oligoglucosides. These materials are also known generically as "alkyl polyglycosides" (APG). The alk(en)yl oligoglycosides according to the invention correspond to formula (II):

[0087] RaO[G]P(II) wherein Rais an alkyl or alkenyl radical having from 6 to 22 carbon atoms, G is a sugar unit having 5 or 6 carbon atoms and p is a number from 1 to 10. The index p in general formula (III) indicates the degree of oligomerisation (DP degree), i.e. the distribution of mono- and oligoglycosides, and is a number of 1 to 10. Whereas p in a given compound must always be an integer and, above all, may assume a value of 1 to 6, the value p for a certain alkyl oligoglycoside is an analytically determined calculated quantity which is mostly a broken number. Alk(en)yl oligoglycosides having an average degree of oligomerisation p of 1 .1 to 3.0 are preferably used. Alk(en)yl oligoglycosides having a degree of oligomerisation below 1.7 and, more particularly, between 1 .2 and 1 .4 are preferred from the applicational point of view. The alkyl or alkenyl radical Ramay be derived from primary alcohols containing 4 to 22 and preferably 8 to 16 carbon atoms. Typical examples are butanol, caproic alcohol, caprylic alcohol, capric alcohol, undecyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, palmitoleyl alcohol, stearyl alcohol, isostearyl alcohol, oleyl alcohol, elaidyl alcohol, petroselinyl alcohol, arachyl alcohol, gadoleyl alcohol, behenyl alcohol, erucyl alcohol and technical mixtures thereof such as are formed, for example, in the hydrogenation of technical fatty acid methyl esters or in the hydrogenation of aldehydes from Roelen’s oxo synthesis. Alkyl oligoglucosides based on hydrogenated Ca-Ci6 coconut oil alcohol having a DP of 1 to 3 are preferred. Also suitable are alkoxylation products of alkyl oligoglucosides, for example adducts of 1 to 10 moles ethylene oxide and / or 1 to 5 moles propylene oxide to Ca-Cio or C12-C18 alkyl oligoglucoside having a DP between 1 .2 and 1 .4.

[0088] In various embodiments, the emulsifier or surfactant is an alkoxylated vegetable oil. Suitable emulsifiers are castor oil, rape seed oil, soy bean oil ethoxylated with 3 to 80 moles ethylene oxide (Agnique CSO 35, Agnique SBO 10, Agnique SBO 60)).

[0089] In various embodiments, the emulsifier or surfactant is an alkoxylated copolymer. Typical copolymers are ethoxylated and propoxylated block and / or random polymers of C2-22 linear or branched alcohols.

[0090] In various embodiments, the emulsifier can be a miscellaneous emulsifier. Typical anionic emulsifiers are for example alkylbenzene sulfonates like dodecylbenzene sulfonate salts (e.g. Agnique® ABS 60 C or65C), di-octyl sulfosuccinates or anionic polymers like polyacrylates. Other suitable emulsifiers are zwitterionic surfactants. Zwitterionic surfactants are surface-active compounds which contain at least one quaternary ammonium group and at least one carboxylate and one sulfonate group in the molecule. Particularly suitable zwitterionic surfactants are the so-called betaines such as the N-alkyl-N,N-dimethyl ammonium glycinates, for example cocoalkyl dimethyl ammonium glycinate, N-acylaminopropyl-N,N-dimethyl ammonium glycinates, for example cocoacylaminopropyl dimethyl ammonium glycinate, and 2-alkyl-3- carboxymethyl-3-hydroxyethyl imidazolines containing 8 to 18 carbon atoms in the alkyl or acyl group and cocoacylaminoethyl hydroxyethyl carboxymethyl glycinate. The fatty acid amide derivative known under the CTFA name of Cocamidopropyl Betaine is particularly preferred. Ampholytic surfactants are also suitable emulsifiers. Ampholytic surfactants are surface-active compounds which, in addition to a Cs / is alkyl or acyl group, contain at least one free amino group and at least one -COOH- or -SO3H- group in the molecule and which are capable of forming inner salts. Examples of suitable ampholytic surfactants are N-alkyl glycines, N-alkyl propionic acids, N-alkylaminobutyric acids, N-alkyliminodipropionic acids, N-hydroxyethyl-N- alkylamidopropyl glycines, N-alkyl taurines, N-alkyl sarcosines, 2-alkylaminopropionic acids and alkylaminoacetic acids containing around 8 to 18 carbon atoms in the alkyl group. Particularly preferred ampholytic surfactants are N-cocoalkylaminopropionate, cocoacylaminoethyl aminopropionate and C12 / 18 acyl sarcosine.

[0091] In various embodiments, the emulsifier or surfactant is selected from non-ionic or anionic surfactants, and mixtures thereof, optionally, castor oil ethoxylates, fatty acid alkoxylates, alcohol alkoxylates, alkyl amine alkoxylates, polyether copolymers, sorbitan ethoxylate esters, aromatic alcohol alkoxylates, alkylbenzene sulfonates or (di-octyl) sulfosuccinates, or mixtures thereof.

[0092] In various embodiments, the at least one emulsifier or surfactant is a castor oil ethoxylate or an alkylbenzene sulfonate or a mixture thereof.

[0093] In addition to these components, the composition may further comprise other adjuvants conventionally used for agrochemical formulations, the choice of the adjuvants depending on the specific use form, the type of formulation or the active substance. Examples of suitable adjuvants are surface-active substances (such as solubilizers, protective colloids, wetters and tackifiers), retention agents, wetting agents, spreaders, uptake enhancers, spray drift controllers, crystallization inhibitors, organic and inorganic thickeners, bactericides, antifreeze agents, antifoams, optionally colorants and adhesives (for example for the treatment of seed).

[0094] In various embodiments, the composition comprises the at least one compound to be tested, for example a known or suspected penetration enhancing adjuvant for agrochemical formulations, which may also be selected from the above-listed potential ingredients of the composition, as well as an agrochemical active. Such a combination may allow determining and quantifying the penetration enhancing properties of the at least one compound with respect to the agrochemical active. Such measurement may include determining the penetration of the active into and / or through the wax layer in the presence and absence of the at least one compound to allow determining its influence on penetration, for example on penetration speed and depth. Such testing may allow to identify combinations of adjuvant and active that work particularly well with respect to their penetration properties in that their penetration properties are improved. In view of the multitude of actives used and known in the field which may vary widely with respect to their physicochemical properties, such as solubility etc., certain actives may work well together with certain adjuvants but not others. The described method allows to identify combinations that complement each other well with respect to the desired penetration properties.

[0095] The term “agrochemical active” refers to a substance that confers a desirable biological activity to the composition. Typically, the agrochemical active is a pesticide. Agrochemical actives are typically selected from fungicides, insecticides, nematicides, herbicides, safeners, nitrification inhibitors, urease inhibitors, plant growth regulators, micronutrients, biopesticides and / or growth regulators. In one embodiment, the agrochemical active is an insecticide. In another embodiment, the agrochemical active is a herbicide. In a further embodiment, the agrochemical active is a fungicide.

[0096] A pesticide is generally a chemical or biological agent (such as pesticidal active ingredient, compound, composition, virus, bacterium, antimicrobial, or disinfectant) that through its effect deters, incapacitates, kills or otherwise discourages pests. Target pests can include insects, plant pathogens, weeds, mollusks, birds, mammals, fish, nematodes (roundworms), and microbes that destroy property, cause nuisance, spread disease or are vectors for disease. The term “pesticide” includes also plant growth regulators that alter the expected growth, flowering, or reproduction rate of plants; defoliants that cause leaves or other foliage to drop from a plant, usually to facilitate harvest; desiccants that promote drying of living tissues, such as unwanted plant tops; plant activators that activate plant physiology for defense of against certain pests; safeners that reduce unwanted herbicidal action of pesticides on crop plants; and plant growth promoters that affect plant physiology e.g. to increase plant growth, biomass, yield or any other quality parameter of the harvestable goods of a crop plant.

[0097] The following lists of pesticides that are suitable for use in compositions of the invention, is intended to illustrate the possible combinations but does not limit them: A) Respiration inhibitors inhibitors of complex III at Qosite: azoxystrobin, coumethoxystrobin, coumoxystrobin, dimoxystrobin, enestroburin, fenaminstrobin, fenoxystrobin / flufenoxystrobin, fluoxastrobin, kresoxim-methyl, mandestrobin, metominostrobin, orysastrobin, picoxystrobin, pyraclostrobin, pyrametostrobin, pyraoxystrobin, trifloxystrobin, pyribencarb, triclopyricarb / chlorodincarb, famoxadone, fenamidone, pyriminostrobin, bifujunzhi, metyltetraprole; inhibitors of complex III at Qi site: cyazofamid, amisulbrom, fenpicoxamid, florylpicoxamid, metarylpicoxamid; inhibitors of complex II: benodanil, benzovindiflupyr, bixafen, boscalid, carboxin, fenfuram, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, mepronil, oxycarboxin, penflufen, penthiopyrad, pydiflumetofen, pyraziflumid, sedaxane, tecloftalam, thifluzamide, inpyrfluxam, pyrapropoyne, fluindapyr, isoflucypram, cyclobutrifluram; other respiration inhibitors: diflumetorim; nitrophenyl derivates: binapacryl, dinobuton, dinocap, fluazinam, meptyldinocap; ferimzone; organometal compounds: fentin salts, e.g. fentin-acetate, fentin chloride or fentin hydroxide; silthiofam; quinone outside inhibitor stigmatellin binding type: ametoctradin.

[0098] B) Sterol biosynthesis inhibitors (SBI fungicides)

[0099] C14 demethylase inhibitors: triazoles: azaconazole, bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole, diniconazole-M, epoxiconazole, fenbuconazole, fluquinconazole, flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, oxpoconazole, paclobutrazole, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol, triticonazole, uniconazole , fluoxytioconazole, ipfentrifluconazole, mefentrifluconazole; imidazoles: imazalil, pefurazoate, prochloraz, triflumizol; pyrimidines, pyridines, piperazines: fenarimol, pyrifenox, triforine;

[0100] Delta14-reductase inhibitors: aldimorph, dodemorph, dodemorph-acetate, fenpropimorph, tridemorph, fenpropidin, piperalin, spiroxamine;

[0101] Inhibitors of 3-keto reductase: fenhexamid, fenpyrazamine; other Sterol biosynthesis inhibitors: chlorphenomizole.

[0102] C) Nucleic acid synthesis inhibitors

[0103] RNA polymerase I inhibitors: benalaxyl, benalaxyl-M, kiralaxyl, metalaxyl, metalaxyl-M, ofurace, oxadixy I; other nucleic acid synthesis inhibitors: hymexazole, octhilinone, oxolinic acid, bupirimate, 5- fluorocytosine, ipflufenoquin, quinofumelin.

[0104] D) Inhibitors of cell division and cytoskeleton

[0105] - tubulin polymerization inhibitors: benomyl, carbendazim, fuberidazole, thiabendazole, thiophanate- methyl, pyridachlometyl; other cell division inhibitors: diethofencarb, ethaboxam, pencycuron, fluopicolide, zoxamide, metrafenone, pyriofenone, phenamacril, fluopimomide. E) Inhibitors of amino acid and protein synthesis methionine synthesis inhibitors: cyprodinil, mepanipyrim, pyrimethanil; protein synthesis inhibitors: blasticidin-S, kasugamycin, kasugamycin hydrochloride-hydrate, mildiomycin, streptomycin, oxytetracycline.

[0106] F) Signal transduction inhibitors

[0107] MAP I histidine kinase inhibitors: fluoroimid, iprodione, procymidone, vinclozolin, fludioxonil; mechanism unknown: quinoxyfen, proquinazid.

[0108] G) Lipid and membrane synthesis inhibitors

[0109] Phospholipid biosynthesis inhibitors: edifenphos, iprobenfos, pyrazophos, isoprothiolane; lipid peroxidation: dicloran, quintozene, tecnazene, tolclofos-methyl, biphenyl, chloroneb, etridiazole; compounds affecting cell membrane permeability and fatty acides: propamocarb; inhibitors of oxysterol binding protein: oxathiapiprolin, fluoxapiprolin.

[0110] H) Inhibitors with Multi Site Action inorganic active substances: Bordeaux mixture, copper, copper acetate, copper hydroxide, copper oxychloride, basic copper sulfate, sulfur;

[0111] - thio- and dithiocarbamates: ferbam, mancozeb, maneb, metam, metiram, propineb, thiram, zineb, ziram; organochlorine compounds: anilazine, chlorothalonil, captafol, captan, folpet, dichlofluanid, dichlorophen, hexachlorobenzene, pentachlorphenole and its salts, phthalide, tolylfluanid; guanidines and others: guanidine, dodine, dodine free bas, guazatine, guazatine-acetate, iminoctadine, iminoctadine-triacetate, iminoctadine-tris(albesilate), dithianon, fluoroimide, methasulfocarb, chinomethionat.

[0112] I) Cell wall synthesis inhibitors inhibitors of glucan synthesis: validamycin, polyoxin B; melanin synthesis inhibitors: pyroquilon, tricyclazole, carpropamid, dicyclomet, fenoxanil, tolprocarb; cellulose synthase inhibitors: dimethomorph, flumorph, mandipropamid, pyrimorph, benthiavalicarb, iprovalicarb, valifenalate.

[0113] J) Plant defense inducers acibenzolar-S-methyl, probenazol, isotianil, tiadinil, prohexadione-calcium; phosphonates: fosetyl, fosetyl-aluminum, phosphorous acid and its salts, calcium phosphonate, potassium phosphonate, potassium or sodium bicarbonate, dichlobentiazox.

[0114] K) Unknown mode of action bronopol, cyflufenamid, cymoxanil, dazomet, debacarb, diclocymet, diclomezine, difenzoquat, difenzoquat-methylsulfate, diphenylamin, fenitropan, fenpyrazamine, flumetover, flumetylsulforim, flusulfamide, flutianil, harpin, nitrapyrin, nitrothal-isopropyl, oxin-copper, seboctylamine, tebufloquin, tecloftalam, triazoxide, pyrisoxazole, benziothiazolinone, bromothalonil, aminopyrifen, flufenoxadiazam.

[0115] L) Biopesticides

[0116] L1) Microbial pesticides with fungicidal, bactericidal, viricidal and / or plant defense activator activity: Ampelomyces quisqualis, Aspergillus flavus, Aureobasidium pullulans, Bacillus altitudinis, B. amyloliquefaciens, B. amyloliquefaciens ssp. plantarum (also referred to as B. velezensis), B. megaterium, B. mojavensis, B. mycoides, B. pumilus, B. simplex, B. solisalsi, B. subtilis, B. subtilis var. amyloliquefaciens, B. velezensis, Candida oleophila, C. saitoana, Clavibacter michiganensis (bacteriophages), Coniothyrium minitans, Cryphonectria parasitica, Cryptococcus albidus, Dilophosphora alopecuri, Fusarium oxysporum, Clonostachys rosea f. catenulate (also named Gliocladium catenulatum), Gliocladium roseum, Lysobacter antibioticus, L. enzymogenes, Metschnikowia fructicola, Microdochium dimerum, Microsphaeropsis ochracea, Muscodor albus, Paenibacillus alvei, Paenibacillus epiphyticus, P. polymyxa, Pantoea vagans, Penicillium bilaiae, Phlebiopsis gigantea, Pseudomonas sp., Pseudomonas chloraphis, Pseudozyma flocculosa, Pichia anomala, Pythium oligandrum, Sphaerodes mycoparasitica, Streptomyces griseoviridis, S. lydicus, S. violaceusniger, Talaromyces flavus, Trichoderma asperelloides, T. asperellum, T. atroviride, T. fertile, T. gamsii, T. harmatum, T. harzianum, T. polysporum, T. stromaticum, T. virens, T. viride, Typhula phacorrhiza, Ulocladium oudemansii, Verticillium dahlia, zucchini yellow mosaic virus (avirulent strain).

[0117] L2) Biochemical pesticides with fungicidal, bactericidal, viricidal and / or plant defense activator activity: harpin protein, Reynoutria sachalinensis extract.

[0118] L3) Microbial pesticides with insecticidal, acaricidal, molluscidal and / or nematicidal activity: Agrobacterium radiobacter, Bacillus cereus, B. firmus, B. thuringiensis, B. thuringiensis ssp. aizawai, B. t. ssp. israelensis, B. t. ssp. galleriae, B. t. ssp. kurstaki, B. t. ssp. tenebrionis, Beauveria bassiana, B. brongniartii, Burkholderia spp., Chromobacterium subtsugae, Cydia pomonella granulovirus, Cryptophlebia leucotreta granulovirus, Flavobacterium spp., Helicoverpa armigera nucleopolyhedrovirus, Helicoverpa zea nucleopolyhedrovirus, Helicoverpa zea single capsid nucleopolyhedrovirus, Heterorhabditis bacteriophora, Isaria fumosorosea, Lecanicillium longisporum, L. muscarium, Metarhizium anisopliae, M. anisopliae var. anisopliae, M. anisopliae var. acridum, Nomuraea rileyi, Paecilomyces fumosoroseus, P. lilacinus, Paenibacillus popilliae, Pasteuria spp., P. nishizawae, P. penetrans, P. ramosa, P. thornea, P. usgae, Pseudomonas fluorescens, Spodoptera littoralis nucleopolyhedrovirus, Steinernema carpocapsae, S. feltiae, S. kraussei, Streptomyces galbus, S. microflavus.

[0119] L4) Biochemical pesticides with insecticidal, acaricidal, molluscidal, pheromone and / or nematicidal activity: L-carvone, citral, (E,Z)-7,9-dodecadien-1-yl acetate, ethyl formate, (E,Z)-2,4-ethyl decadienoate (pear ester), (Z,Z,E)-7,11 ,13-hexadecatrienal, heptyl butyrate, isopropyl myristate, lavanulyl senecioate, cis-jasmone, 2-methyl 1-butanol, methyl eugenol, methyl jasmonate, (E,Z)-2,13-octadecadien-1-ol, (E,Z)- 2,13-octadecadien-1-ol acetate, (E,Z)-3,13-octadecadien-1-ol, (R)-1-octen-3-ol, pentatermanone, (E,Z,Z)-3,8,11 -tetradecatrienyl acetate, (Z,E)-9,12-tetradecadien-1-yl acetate, (Z)-7-tetradecen-2-one, (Z)- 9-tetradecen-1-yl acetate, (Z)-11-tetradecenal, (Z)-11-tetradecen-1-ol, extract of Chenopodium ambrosiodes, Neem oil, Quillay extract.

[0120] L5) Microbial pesticides with plant stress reducing, plant growth regulator, plant growth promoting and / or yield enhancing activity: Azospirillum amazonense, A. brasilense, A. lipoferum, A. irakense, A. halopraeferens, Bradyrhizobium spp., B. elkanii, B. japonicum, B. liaoningense, B. lupini, Delftia acidovorans, Glomus intraradices, Mesorhizobium spp., Rhizobium leguminosarum bv. phaseoli, R. I. bv. tri foil! , R. I. bv. viciae, R. tropic!, Sinorhizobium meliloti.

[0121] O) Insecticides from classes 0.1 to 0.31

[0122] 0.1 Acetylcholine esterase (AChE) inhibitors: aldicarb, alanycarb, bendiocarb, benfuracarb, butocarboxim, butoxycarboxim, carbaryl, carbofuran, carbosulfan, ethiofencarb, fenobucarb, formetanate, furathiocarb, isoprocarb, methiocarb, methomyl, metolcarb, oxamyl, pirimicarb, propoxur, thiodicarb, thiofanox, trimethacarb, XMC, xylylcarb, triazamate; acephate, azamethiphos, azinphos-ethyl, azinphosmethyl, cadusafos, chlorethoxyfos, chlorfenvinphos, chlormephos, chlorpyrifos, chlorpyrifos-methyl, coumaphos, cyanophos, demeton-S-methyl, diazinon, dichlorvos / DDVP, dicrotophos, dimethoate, dimethylvinphos, disulfoton, EPN, ethion, ethoprophos, famphur, fenamiphos, fenitrothion, fenthion, fosthiazate, heptenophos, imicyafos, isofenphos, isopropyl O-(methoxyaminothio-phosphoryl) salicylate, isoxathion, malathion, mecarbam, methamidophos, methidathion, mevinphos, monocrotophos, naled, omethoate, oxydemeton-methyl, parathion, parathion-methyl, phenthoate, phorate, phosalone, phosmet, Phosphamidon, phoxim, pirimiphos-methyl, profenofos, propetamphos, prothiofos, pyraclofos, pyridaphenthion, quinalphos, sulfotep, tebupirimfos, temephos, terbufos, tetrachlorvinphos, thiometon, triazophos, trichlorfon, vamidothion.

[0123] 0.2 GABA-gated chloride channel antagonists: endosulfan, chlordane; ethiprole, fipronil, flufiprole, pyrafluprole, pyriprole.

[0124] 0.3 Sodium channel modulators: acrinathrin, allethrin, d-cis-trans allethrin, d-trans allethrin, bifenthrin, kappa-bifenthrin, bioallethrin, bioallethrin S-cylclopentenyl, bioresmethrin, cycloprothrin, cyfluthrin, beta- cyfluthrin, cyhalothrin, lambda-cyhalothrin, gamma-cyhalothrin, cypermethrin, alpha-cypermethrin, beta- cypermethrin, theta-cypermethrin, zeta-cypermethrin, cyphenothrin, deltamethrin, empenthrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, flumethrin, tau-fluvalinate, halfenprox, heptafluthrin, imiprothrin, meperfluthrin, metofluthrin, momfluorothrin, epsilon-momfluorothrin, permethrin, phenothrin, prallethrin, profluthrin, pyrethrin (pyrethrum), resmethrin, silafluofen, tefluthrin, kappa-tefluthrin, tetramethylfluthrin, tetramethrin, tralomethrin, transfluthrin; DDT, methoxychlor.

[0125] 0.4 Nicotinic acetylcholine receptor (nAChR) agonists: acetamiprid, clothianidin, cycloxaprid, dinotefuran, imidacloprid, nitenpyram, thiacloprid, thiamethoxam; nicotine; sulfoxaflor, flupyradifurone, triflumezopyrim, fenmezoditiaz, flupyrimin. 0.5 Nicotinic acetylcholine receptor allosteric activators: spinosad, spinetoram.

[0126] 0.6 Chloride channel activators: abamectin, emamectin benzoate, ivermectin, lepimectin, milbemectin.

[0127] 0.7 Juvenile hormone mimics: hydroprene, kinoprene, methoprene; fenoxycarb, pyriproxyfen.

[0128] 0.8 miscellaneous non-specific (multi-site) inhibitors: methyl bromide and other alkyl halides; chloropicrin, sulfuryl fluoride, borax, tartar emetic.

[0129] 0.9 Chordotonal organ TRPV channel modulators: afidopyropen, pymetrozine, pyrifluquinazon.

[0130] 0.10 Mite growth inhibitors: clofentezine, hexythiazox, diflovidazin; etoxazole.

[0131] 0.11 Microbial disruptors of insect midgut membranes: Bacillus thuringiensis, B. sphaericus and the insecticidal proteins they produce: Bacillus thuringiensis subsp. israelensis, B. sphaericus, B. thuringiensis subsp. aizawai, B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. tenebrionis, the Bt crop proteins: CrylAb, CrylAc, CrylFa, Cry2Ab, mCry3A, Cry3Ab, Cry3Bb, Cry34 / 35Ab1.

[0132] 0.12 Inhibitors of mitochondrial ATP synthase: diafenthiuron; azocyclotin, cyhexatin, fenbutatin oxide, propargite, tetradifon.

[0133] 0.13 Uncouplers of oxidative phosphorylation via disruption of the proton gradient: chlorfenapyr, DNOC, sulfluramid.

[0134] 0.14 Nicotinic acetylcholine receptor (nAChR) channel blockers: bensultap, cartap hydrochloride, thiocyclam, thiosultap sodium.

[0135] 0.15 Inhibitors of the chitin biosynthesis type 0: bistrifluron, chlorfluazuron, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, teflubenzuron, triflumuron.

[0136] 0.16 Inhibitors of the chitin biosynthesis type 1 : buprofezin.

[0137] 0.17 Moulting disruptors: cyromazine.

[0138] 0.18 Ecdyson receptor agonists: methoxyfenozide, tebufenozide, halofenozide, fufenozide, chromafenozide.

[0139] 0.19 Octopamin receptor agonists: amitraz.

[0140] 0.20 Mitochondrial complex III electron transport inhibitors: hydramethylnon, acequinocyl, fluacrypyrim, bifenazate. 0.21 Mitochondrial complex I electron transport inhibitors: fenazaquin, fenpyroximate, pyrimidifen, pyridaben, tebufenpyrad, tolfenpyrad; rotenone.

[0141] 0.22 Voltage-dependent sodium channel blockers: indoxacarb, metaflumizone.

[0142] 0.23 Inhibitors of the of acetyl CoA carboxylase: spirodiclofen, spiromesifen, spirotetramat, spiropidion, spirobudifen, spidoxamat.

[0143] 0.24 Mitochondrial complex IV electron transport inhibitors: aluminium phosphide, calcium phosphide, phosphine, zinc phosphide, cyanide.

[0144] 0.25 Mitochondrial complex II electron transport inhibitors: cyenopyrafen, cyflumetofen, cyetpyrafen, pyflubumide.

[0145] 0.26 Ryanodine receptor-modulators: chlorantraniliprole, cyantraniliprole, cyclaniliprole, flubendiamide, fluchlodiniliprole; tetrachlorantraniliprole; tetraniliprole; tiorantraniliprole; cyhalodiamide.

[0146] 0.27 Chordotonal organ modulators: flonicamid.

[0147] 0.28 GABA-gated chloride channel allosteric modulators: broflanilide, fluxametamide, isocycloseram.

[0148] 0.29 Calcium-activated potassium channel modulators: acynonapyr.

[0149] 0.30 Mitochondrial complex III electron transport inhibitors at Qi site: flometoquin.

[0150] 0.31 Insecticidal compounds of unknown or uncertain mode of action: afoxolaner, azadirachtin, amidoflumet, benzoximate, bromopropylate, chinomethionat, cryolite, cyproflanilid, dicloromezotiaz, dicofol, dimpropyridaz, flufenerim, flometoquin, fluensulfone, fluhexafon, fluopyram, fluralaner, metaldehyde, metoxadiazone, piperonyl butoxide, pyridalyl, tioxazafen, trifluenfuronate, umifoxolaner, actives on basis of Bacillus firmus (Votivo); fluazaindolizine; tyclopyrazoflor; sarolaner, lotilaner; benzpyrimoxan; tigolaner; oxazosulfyl; cyproflanilide, nicofluprole; indazapyroxamet; flupentiofenox; cyclobutrifluram.

[0151] P) Herbicides from the classes P1 to P15

[0152] P1) lipid biosynthesis inhibitors:

[0153] - ACC-herbicides: alloxydim, alloxydim-sodium, butroxydim, clethodim, clodinafop, clodinafop-propargyl, cycloxydim, cyhalofop, cyhalofop-butyl, diclofop, diclofop-methyl, fenoxaprop, fenoxaprop-ethyl, fenoxaprop-P, fenoxaprop-P-ethyl, fluazifop, fluazifop-butyl, fluazifop-P, fluazifop-P-butyl, haloxyfop, haloxyfop-methyl, haloxyfop-P, haloxyfop-P-methyl, metamifop, pinoxaden, profoxydim, propaquizafop, quizalofop, quizalofop-ethyl, quizalofop-tefuryl, quizalofop-P, quizalofop-P-ethyl, quizalofop-P-tefuryl, sethoxydim, tepraloxydim, tralkoxydim;

[0154] - non ACC herbicides: benfuresate, butylate, cycloate, dalapon, dimepiperate, EPTC, esprocarb, ethofumesate, flupropanate, molinate, orbencarb, pebulate, prosulfocarb, TCA, thiobencarb, tiocarbazil, triallate and vernolate.

[0155] P2) ALS inhibitors:

[0156] - sulfonylureas: amidosulfuron, azimsulfuron, bensulfuron, bensulfuron-methyl, chlorimuron, chlorimuron- ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, iodosulfuron-methyl- sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, metazosulfuron, metsulfuron, metsulfuron- methyl, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, primisulfuron-methyl, propyrisulfuron, prosulfuron, pyrazosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron, thifensulfuron-methyl, triasulfuron, tribenuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron , triflusulfuron-methyl and tritosulfuron;

[0157] - imidazolinones: imazamethabenz, imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin and imazethapyr;

[0158] - triazolopyrimidine herbicides and sulfonanilides: cloransulam, cloransulam-methyl, diclosulam, flumetsulam, florasulam, metosulam, penoxsulam, pyrimisulfan and pyroxsulam;

[0159] - pyrimidinylbenzoates: bispyribac, bispyribac-sodium, pyribenzoxim, pyriftalid, pyriminobac, pyriminobac-methyl, pyrithiobac, pyrithiobac-sodium;

[0160] - sulfonylaminocarbonyl-triazolinone herbicides: flucarbazone, flucarbazone-sodium, propoxycarbazone, propoxycarbazone-sodium, thiencarbazone and thiencarbazone-methyl;

[0161] - and triafamone.

[0162] P3) photosynthesis inhibitors: amicarbazone, inhibitors of the photosystem II, triazine herbicides, including of chlorotriazine, triazinones, triazindiones, methylthiotriazines and pyridazinones such as ametryn, atrazine, chloridazone, cyanazine, desmetryn, dimethametryn, hexazinone, metribuzin, prometon, prometryn, propazine, simazine, simetryn, terbumeton, terbuthylazin, terbutryn and trietazin, aryl urea such as chlorobromuron, chlorotoluron, chloroxuron, dimefuron, diuron, fluometuron, isoproturon, isouron, linuron, metamitron, methabenzthiazuron, metobenzuron, metoxuron, monolinuron, neburon, siduron, tebuthiuron and thiadiazuron, phenyl carbamates such as desmedipham, karbutilat, phenmedipham, phenmedipham-ethyl, nitrile herbicides such as bromofenoxim, bromoxynil and its salts and esters, ioxynil and its salts and esters, uraciles such as bromacil, lenacil and terbacil, and bentazon and bentazon-sodium, pyridate, pyridafol, pentanochlor and propanil and inhibitors of the photosystem I such as diquat, diquat-dibromide, paraquat, paraquat-dichloride and paraquat-dimetilsulfate.

[0163] P4) protoporphyrinogen-IX oxidase inhibitors: acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, chlorphthalim, cinidon-ethyl, cyclopyranil, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraflufen-ethyl, saflufenacil, sulfentrazone, thidiazimin, tiafenacil, trifludimoxazin, epyrifenacil.

[0164] P5) bleacher herbicides:

[0165] - PDS inhibitors: beflubutamid, diflufenican, fluridone, flurochloridone, flurtamone, norflurazon, picolinafen, rimisoxafen;

[0166] - HPPD inhibitors: benzobicyclon, benzofenap, bicyclopyrone, clomazone, fenquinotrione, isoxaflutole, mesotrione, oxotrione, pyrasulfotole, pyrazolynate, pyrazoxyfen, sulcotrione, tefuryltrione, tembotrione, tolpyralate, topramezone, bipyrazone, fenpyrazone, cypyrafluone, tripyrasulfone, benquitrione, dioxopyritrione;

[0167] - bleacher, unknown target: aclonifen, amitrole flumeturon, bixlozone.

[0168] P6) EPSP synthase inhibitors: glyphosate, glyphosate-isopropylammonium, glyposate-potassium and glyphosate-trimesium (sulfosate).

[0169] P7) glutamine synthase inhibitors: bilanaphos (bialaphos), bilanaphos-sodium, glufosinate, glufosinate-P and glufosinate-ammonium.

[0170] P8) DHP synthase inhibitors: asulam.

[0171] P9) mitosis inhibitors:

[0172] - group K1 : dinitroanilines: benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine and trifluralin; phosphoramidates: amiprophos, amiprophos-methyl, and butamiphos; benzoic acid herbicides: chlorthal, chlorthal-dimethyl; pyridines: dithiopyr and thiazopyr; benzamides: propyzamide and tebutam;

[0173] - group K2: carbetamide, chlorpropham, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M- isopropyl, flamprop-M-methyl and propham.

[0174] P10) VLCFA inhibitors:

[0175] - chloroacetamides: acetochlor, alachlor, amidochlor, butachlor, dimethachlor, dimethenamid, dimethenamid-P, metazachlor, metolachlor, metolachlor-S, pethoxamid, pretilachlor, propachlor, propisochlor and thenylchlor,

[0176] - oxyacetanilides: flufenacet and mefenacet;

[0177] - acetanilides: diphenamid, naproanilide, napropamide and napropamide-M;

[0178] - tetrazolinones: fentrazamide;

[0179] - other herbicides: anilofos, cafenstrole, fenoxasulfone, ipfencarbazone, piperophos, pyroxasulfone, dimesulfazet and isooxazoline.

[0180] P11) cellulose biosynthesis inhibitors: chlorthiamid, dichlobenil, flupoxam, indaziflam, isoxaben, triaziflam. P12) decoupler herbicides: dinoseb, dinoterb and DNOC and its salts.

[0181] P13) auxinic herbicides:

[0182] 2,4-D and its salts and esters such as clacyfos, 2,4-DB and its salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts such as aminopyralid-dimethylammonium, aminopyralid- tris(2-hydroxypropyl)ammonium and its esters, benazolin, benazolin-ethyl, chloramben and its salts and esters, clomeprop, clopyralid and its salts and esters, dicamba and its salts and esters, dichlorprop and its salts and esters, dichlorprop-P and its salts and esters, flopyrauxifen, fluroxypyr, fluroxypyr-butometyl, fluroxypyr-meptyl, halauxifen and its salts and esters; MCPA and its salts and esters, MCPA-thioethyl, MCPB and its salts and esters, mecoprop and its salts and esters, mecoprop-P and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac, TBA (2,3,6) and its salts and esters, triclopyr and its salts and esters, florpyrauxifen, florpyrauxifen-benzyl and 4-amino-3-chloro-5-fluoro-6-(7-fluoro-1 / 7- indol-6-yl)picolinic acid.

[0183] P14) auxin transport inhibitors: diflufenzopyr, diflufenzopyr-sodium, naptalam and naptalam-sodium.

[0184] P15) other herbicides: bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, cyclopyrimorate and its salts and esters, dalapon, dazomet, difenzoquat, difenzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, etobenzanid, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, maleic hydrazide, mefluidide, metam, methiozolin, methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, tetflupyrolimet, tridiphane.

[0185] Q) Safeners

[0186] (quinolin-8-oxy)acetic acids, 1-phenyl-5-haloalkyl-1 H-1 ,2,4-triazol-3-carboxylic acids, 1-phenyl-4,5- dihydro-5-alkyl-1 H-pyrazol-3,5-dicarboxylic acids, 4,5-dihydro-5,5-diaryl-3-isoxazol carboxylic acids, dichloroacetamides, alpha-oximinophenylacetonitriles, acetophenonoximes, 4,6-dihalo-2- phenylpyrimidines, A / -[[4-(aminocarbonyl)phenyl]sulfonyl]-2-benzoic amides, 1 ,8-naphthalic anhydride, 2- halo-4-(haloalkyl)-5-thiazol carboxylic acids, phosphorthiolates and N-alkyl-O-phenylcarbamates and their agriculturally acceptable salts and their agriculturally acceptable derivatives such amides, esters, and thioesters, provided they have an acid group.

[0187] In various embodiments, the composition comprises at least one agrochemical active selected from the group consisting of herbicides, fungicides, and insecticides, and / or at least one plant growth regulator or a combination of any two or more of the afore-mentioned.

[0188] The composition may be in any form, such as a solution, dispersion, emulsion, suspension and the like.

[0189] If the wax layer is placed on a solid support, the contacting is with the wax layer surface opposite to the surface contacting the solid support. The composition may be contacted with the wax layer by any suitable means, including, for example and without limitation, spraying, pouring, dipping, dripping, and the like. In various embodiments, it is sprayed onto the wax layer surface. In various other embodiments, it may be dispensed onto the wax layer by a pipette or syringe or similar device, optionally in an automated mode.

[0190] In various embodiments, contacting is carried out for a predefined period of time, which is generally considered sufficient to allow the desired penetration. Such time period may, in various instances, be up to 24 hours, but typically is in the range of several minutes to several hours. In various embodiments, the contacting may be for any time period in the range of 10 minutes to 22 hours, such as 30 minutes to 20 hours, 1 hour to 12 hours, 2 hours to 8 hours and the like. The time period may be selected depending on the observed penetration speed, i.e. kept relatively short for compounds that penetrate quickly into the wax layer and extended to longer periods of time if penetration is slow.

[0191] In various embodiments, the penetration of the compound into and / or across the model plant wax layer is determined by measuring Nile Red fluorescence, for example by monitoring Nile Red fluorescence over time for increases or decreases in fluorescence, which are indicative of penetration into the wax layer. In various embodiments, an increase in Nile Red fluorescence is considered indicative of penetration into the wax layer, with the level of change being correlated to the concentration of the penetrating agent / compound in the respective part of the wax layer.

[0192] Determination of the penetration properties is generally done by determining Nile Red (local) fluorescence, in particular by monitoring (the change in (local)) Nile Red fluorescence over time. Said determination is preferably done by fluorescence measurements by any suitable means, such as fluorescence microscopy, in particular confocal fluorescence microscopy.

[0193] Nile Red fluorescence can be determined, in various embodiments, at an excitation wavelength of about 561 nm and / or an emission wavelength in the range of about 565 to about 750 nm or, alternatively, excitation at about 488 nm and emission detection at 500 to 700 nm, or, still alternatively, excitation at about 530 nm and emission detection at 540 to 700 nm. It is understood that excitation and emission wavelength are typically different in that the emission is at a determinably longer wavelength than excitation due to the energy loss upon fluorescence. It is further understood that subranges of the above emission ranges can be used for detection.

[0194] For determining the penetration properties of said compound the following measuring options can be used: (1) measuring the change in Nile Red fluorescence intensity, (2) measuring and calculating the Nile Red fluorescence intensity ratio between a first channel in the range of 565 to 645 nm and a second channel in the range of 650 to 745 nm, and / or (3) Nile Red fluorescence lifetime imaging (FLIM).

[0195] The first option measures changes in fluorescence intensity. This determination method is based on the change, typically increase of Nile Red fluorescence, that occurs if the at least one compound penetrates into the wax layer and interferes, , i.e. weakens, with the quenching effect the wax molecules have on the Nile Red fluorescence.

[0196] The second option measures changes in the fluorescence intensity ratio which is a measure for local polarity changes in the wax layer. In the Nile Red fluorescence spectrum, a shift to longer wavelengths is observed in case local polarity increases, i.e. the ratiometric ratio is inversely proportional to polarity. Such polarity increase can be caused by water uptake of the wax film and / or by penetration of the at least one compound into the wax film. This polarity change causes a red shift in the Nile Red fluorescence spectrum which can be determined by measuring the fluorescence intensity ratio between a first shorter wavelength channel and a second longer wavelength channel. The short wavelength channel may be selected to be in the range of about 565 to about 645 nm and the longer wavelength channel between about 650 and about 745 nm, wherein both channels should not overlap. A decrease in the ratio of fluorescence intensity of the first shorter wavelength channel to the second longer wavelength channel is indicative for an increase in polarity in the wax layer. In various embodiments, in particular if aqueous compositions are used, an increase of polarity is observed first, caused by water uptake into the wax layer, which is measurable by a decrease of the fluorescence intensity ratios. This is then followed by an increase in fluorescence intensity ratios caused by a decrease in polarity due to migration / penetration of the compound of interest into the wax layer.

[0197] Fluorescence intensity measurements are typically and in various embodiments made using fluorescence microscopy, in particular confocal fluorescence microscopy, such as an inverted confocal laser-scanning microscope.

[0198] The third option uses FLIM and is based on the quenching of Nile Red fluorescence in the wax layer which causes the initial lifetime to be comparably short. Upon penetration of the at least one compound into the wax layer, the quenching is reduced and fluorescence lifetime increases. This increase is measurable and a means to determine the penetration properties of the at least one compound tested. FLIM measurements are, in various embodiments, made using a fluorescence microscope, such as an inverted confocal laserscanning microscope, coupled with a TCSPC module. Time-correlated single photon counting (TCSPC) is a common technique to measure fluorescence decays in the time domain. In principle, single photon events are detected and their time of arrival is correlated to the laser pulse, which is used for excitation of the sample. By using a pulsed laser with a high repetition rate, this process can be repeated many times so that a photon distribution over the time and the spatial coordinates is built up. On the stage of a laser scanning microscope the arrival time of the photon and the coordinates of the laser beam in the scan area are used to build up a 3-dimensional matrix, in which for each pixel of the scanned image not only the fluorescence intensity, but a fluorescence decay curve is stored. For each photon, the TCSPC module determines the location within the scanning area (x and y) and the time of the photon in the laser pulse sequence (t). These values are used to address a memory in which the detection events are accumulated so that in the memory the distribution of the photon density over x, y and t is built up. The method can be used as a screening method to determine the penetration properties of a multitude of compounds, for example in parallel and optionally in an automated fashion. The method may thus be in a multiplexing format.

[0199] The present invention further relates to the use of Nile Red comprised in a wax layer optionally in a homogeneous distribution for determining the penetration properties of at least one compound into and / or through said wax layer by monitoring the change in Nile Red fluorescence upon contacting with the at least one compound. In such embodiments, the wax layer comprising the Nile Red is provided prior to contacting the at least one compound with said wax layer.

[0200] All embodiments disclosed above in relation to the inventive methods are similarly applicable to such uses.

[0201] The present invention is further also directed to a wax layer or wax film that comprises at least one wax, preferably at least one plant wax, and Nile Red, wherein the Nile Red is optionally homogeneously distributed in the wax layer. Said wax layer or film may be provided on a solid support, such as a microscope slide, for example a glass slide. Such solid support with a wax layer deposited thereon is thus also an embodiment of the present invention. In various embodiments, the solid support is a glass slide and / or not plant material, such as a plant leaf or part thereof.

[0202] All embodiments disclosed above in relation to the methods and uses of the present invention, in particular with respect to the properties of the wax layer, such as its composition, thickness and Nile Red content as well as the characteristics of the solid support are similarly applicable to this aspect of the invention and vice versa.

[0203] The wax films described herein are intended to be used as a model for the plant’s cuticular wax layer. The methods and uses are specifically adapted to simulate a plant’s cuticular wax layer for assessing penetration / permeation properties of various compounds and compositions through the wax layer. It has been found that the results obtained in these models are readily transferable to real plants and may thus help to simulate how transport of an active into the plant cells can be optimized.

[0204] The term "plant", as used herein in various embodiments of the invention, is preferably to be understood as a plant of economic importance and / or men-grown plant. In certain embodiments, the term may also be understood as plants which have no or no significant economic importance. The plant is preferably selected from agricultural, silvicultural, and horticultural (including ornamental) plants. The term also relates to genetically modified plants.

[0205] The term "plant" as used herein further includes all parts of a plant such as germinating seeds, emerging seedlings, plant propagules, herbaceous vegetation as well as established woody plants including all belowground portions (such as the roots) and aboveground portions. In one embodiment, the plant is an agricultural plant. "Agricultural plants" are plants of which a part (e.g. seeds) or all is harvested or cultivated on a commercial scale or which serve as an important source of feed, food, fibers (e.g. cotton, linen), combustibles (e.g. wood, bioethanol, biodiesel, biomass) or other chemical compounds. Preferred agricultural plants are for example cereals, e.g. wheat, rye, barley, triticale, oats, corn, sorghum or rice, beet, e.g. sugar beet or fodder beet; fruits, such as pomes, stone fruits or soft fruits, e.g. apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries; leguminous plants, such as lentils, peas, alfalfa or soybeans; oil plants, such as rape, oilseed rape, canola, linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans; cucurbits, such as squashes, cucumber or melons; fiber plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruits or mandarins; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or paprika; lauraceous plants, such as avocados, cinnamon or camphor; energy and raw material plants, such as corn, soybean, rape, canola, sugar cane or oil palm; tobacco; nuts; coffee; tea; bananas; vines (table grapes and grape juice grape vines); hop; turf; natural rubber plants.

[0206] In a further embodiment, the plant is a horticultural plant. The term "horticultural plants" are to be understood as plants which are commonly used in horticulture, e.g. the cultivation of ornamentals, vegetables and / or fruits. Examples for ornamentals are turf, geranium, pelargonia, petunia, begonia and fuchsia. Examples for vegetables are potatoes, tomatoes, peppers, cucurbits, cucumbers, melons, watermelons, garlic, onions, carrots, cabbage, beans, peas, and lettuce and more preferably from tomatoes, onions, peas, and lettuce. Examples for fruits are apples, pears, cherries, strawberry, citrus, peaches, apricots, and blueberries.

[0207] In a further embodiment, the plant is an ornamental plant. "Ornamental plants" are plants which are commonly used in gardening, e.g. in parks, gardens and on balconies. Examples are turf, geranium, pelargonia, petunia, begonia and fuchsia.

[0208] In another embodiment of the present invention, the plant is a silvicultural plant. The term "silvicultural plant" is to be understood as trees, more specifically trees used in reforestation or industrial plantations. Industrial plantations generally serve for the commercial production of forest products, such as wood, pulp, paper, rubber tree, Christmas trees, or young trees for gardening purposes. Examples for silvicultural plants are conifers, like pines, in particular Pinus spec., fir and spruce, eucalyptus, tropical trees like teak, rubber tree, oil palm, willow (Salix), in particular Salix spec., poplar (cottonwood), in particular Populus spec., beech, in particular Fagus spec., birch, oil palm, and oak.

[0209] The term "genetically modified plants" is to be understood as plants, which genetic material has been modified by the use of recombinant DNA techniques in a way that under natural circumstances it cannot readily be obtained by cross breeding, mutations, or natural recombination. Typically, one or more genes have been integrated into the genetic material of a genetically modified plant in order to improve certain properties of the plant. Such genetic modifications also include but are not limited to targeted post- translational modification of protein(s), oligo- or polypeptides e. g. by glycosylation or polymer additions such as prenylated, acetylated or farnesylated moieties or PEG moieties.

[0210] Plants that have been modified by breeding, mutagenesis or genetic engineering, e.g. have been rendered tolerant to applications of specific classes of herbicides, such as auxin herbicides such as dicamba or 2,4- D; bleacher herbicides such as hydroxylphenylpyruvate dioxygenase (HPPD) inhibitors or phytoene desaturase (PDS) inhibitors; acetolactate synthase (ALS) inhibitors such as sulfonyl ureas or imidazolinones; enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, such as glyphosate; glutamine synthetase (GS) inhibitors such as glufosinate; protoporphyrinogen-IX oxidase inhibitors; lipid biosynthesis inhibitors such as acetyl CoA carboxylase (ACCase) inhibitors; or oxynil (i.e. bromoxynil or ioxynil) herbicides as a result of conventional methods of breeding or genetic engineering, are also covered. Furthermore, plants have been made resistant to multiple classes of herbicides through multiple genetic modifications, such as resistance to both glyphosate and glufosinate or to both glyphosate and a herbicide from another class such as ALS inhibitors, HPPD inhibitors, auxin herbicides, or ACCase inhibitors. These herbicide resistance technologies are e. g. described in Pest Managem. Sci. 61 , 2005, 246; 61 , 2005, 258; 61 , 2005, 277; 61 , 2005, 269; 61 , 2005, 286; 64, 2008, 326; 64, 2008, 332; Weed Sci. 57, 2009, 108; Austral. J. Agricult. Res. 58, 2007, 708; Science 316, 2007, 1185; and references quoted therein. Several cultivated plants have been rendered tolerant to herbicides by conventional methods of breeding (mutagenesis), e.g. Clearfield® summer rape (Canola, BASF SE, Germany) being tolerant to imidazolinones, e. g. imazamox, or ExpressSun® sunflowers (DuPont, USA) being tolerant to sulfonyl ureas, e. g. tribenuron. Genetic engineering methods have been used to render cultivated plants such as soybean, cotton, corn, beets, and rape, tolerant to herbicides such as glyphosate and glufosinate, some of which are commercially available under the trade names RoundupReady® (glyphosate-tolerant, Monsanto, U.S.A.), Cultivance® (imidazolinone tolerant, BASF SE, Germany) and LibertyLink® (glufosinate-tolerant, Bayer CropScience, Germany). The methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, e.g. in the publications mentioned above.

[0211] Furthermore, plants are also covered that are by the use of recombinant DNA techniques capable to synthesize one or more proteins to increase the resistance or tolerance of those plants to bacterial, viral, or fungal pathogens. Examples of such proteins are the so-called “pathogenesis-related proteins” (PR proteins, see, e. g. EP-A 392 225), plant disease resistance genes (e. g. potato cultivars, which express resistance genes acting against Phytophthora infestans derived from the Mexican wild potato Solanum bulbocastanum) or T4-lysozym (e. g. potato cultivars capable of synthesizing these proteins with increased resistance against bacteria such as Erwinia amylvora). The methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, e.g. in the publications mentioned above.

[0212] Furthermore, plants are also covered that are by the use of recombinant DNA techniques capable to synthesize one or more proteins to increase the productivity (e. g. biomass production, grain yield, starch content, oil content or protein content), tolerance to drought, salinity or other growth-limiting environmental factors or tolerance to pests and fungal, bacterial, or viral pathogens of those plants. Furthermore, plants are also covered that contain by the use of recombinant DNA techniques a modified amount of substances of content or new substances of content, specifically to improve human or animal nutrition, e. g. oil crops that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e. g. Nexera® rape, DOW Agro Sciences, Canada).

[0213] Moreover, plants are also covered that contain by the use of recombinant DNA techniques a modified amount of substances of content or new substances of content, specifically to improve raw material production, e. g. potatoes that produce increased amounts of amylopectin (e. g. Amflora® potato, BASF SE, Germany).

[0214] All embodiments and examples described herein for the composition also apply to the uses and methods disclosed herein and vice versa.

[0215] The present invention is further illustrated by the following examples.

[0216] EXAMPLES

[0217] Materials, instruments and methods

[0218] Confocal Laser Scanning Microscopy (CLSM)

[0219] CLSM, the principle of which is schematically shown in Figure 1 , allows optical sectioning, both horizontally and vertically with lateral resolution >200 nm and vertical resolution >400 nm. The imaging contrast can be based on fluorescence (endogenous or exogenous dyes) or reflection / scattering (refractive index differences). Imaging of fast dynamic processes with frame rates up to 30 Hz and laser illumination of the sample from below (inverted setup) or above (upright setup) is possible. Preferably, there are various kinds of fluorescence dyes available: e.g. hydrophilic, hydrophobic, charged, pH-sensitive, water-sensitive or polymer-specific dyes. For further information see https: / / www.microscopyu.com / techniques / confocal_.

[0220] Imaging parameter

[0221] Leica TCS SP5 II inverted confocal laser-scanning microscope (Leica Microsystem Ltd, Germany). Nile Red was excited at 930 nm using a Ti: Sapphire pulsed laser source (680-1080 nm, 80 MHz, 140 fs, Chameleon Vision II, Coherent Inc., Germany) and the emission intensity was recorded at 520-700 nm. Fluorescence intensity measurements were carried out using the objective and excitation / emission wavelengths as listed below. FLIM images were obtained using the same Leica TCS SP5 II microscope with x63 objective coupled with a TCSPC module SPC830 (Becker&Hickl, Germany) and internal FLIM detector PMH-100 (Becker&Hickl, Germany), synchronised to the Ti: Sapphire pulsed laser.

[0222] Excitation wavelength:

[0223] 405 nm (Coumarin 343) 488 nm (NBD-Pz / Fluorol Yellow)

[0224] 561 nm (Nile Red)

[0225] Emission wavelength:

[0226] 410-530 nm (Coumarin 343)

[0227] 500-550 nm (NBD-Pz / Fluorol Yellow)

[0228] 640-750 nm (Nile Red)

[0229] Ratiometric detection (Nile Red):

[0230] Channel 1 : 565-645 nm

[0231] Channel 2: 650-745 nm

[0232] Objective:

[0233] 63 x 1.40 OIL

[0234] Image Size:

[0235] 70 x 70 pm

[0236] 246 x 246 m

[0237] Scan time / line:

[0238] 400 Hz

[0239] Scan mode: xzy, xyz, xzt

[0240] Sample Materials

[0241] Wax: Carnauba wax (cuticle wax) from Copernicia prunifera and sold by Alfa Aesar, having a melting point of 82-86 °C, a hydrocarbon length of C28-34, an ester content of >80 % and high crystallinity;

[0242] Adjuvants: Plurafac® LF431 ; Plurafac® LF1300; Agnique® SBO10; Lutensol® XP80.

[0243] Preparation of wax layer

[0244] 500 ppm Nile Red was dissolved in a carnauba wax solution (10 wt.-% wax in CHCh at 70 °C). For preparation of a thin wax layer (~ 10 pm) the solution was heated up to 80 °C and coated onto a glass slide by a doctor blade (heated up slightly in order to get a uniform layer thickness). Afterwards the wax layer was heated up to 120 °C for 2 h to release residual chloroform from the layer.

[0245] Test compositions

[0246] Different adjuvants to be tested were dissolved in hard water (pH: 6.5-7) in a concentration of 1000 ppm and applied directly on top of the wax layer.

[0247] In certain test compositions as a model-like active ingredient 500 ppm coumarin 343 was added. Example 1 : Fluorescence intensity and FLIM analysis

[0248] Carnauba wax films stained with 330 ppm Nile Red were analyzed by transmission microscopy (Figure 3a), fluorescence intensity measurements (Figure 3b-d) and FLIM imaging (Figure 3g-i).

[0249] Figures 3b and 3g show measurements / imaging of the dry wax. Figures 3c and 3h show the same wax layer after 60 minutes exposure to an aqueous solution of 1000 ppm Plurafac® LF431 and Figures 3d and 3i after 60 minutes exposure to neat Plurafac® LF431 .

[0250] Figure 3e shows the averaged intensities per frame (N=6 for each condition) vs. averaged lifetime obtained from Figure 3j; the line is given as a visual aid. Figure 3f shows fluorescence spectra and Figure 3j ti histograms obtained from samples presented in (g-i). All data were obtained after excitation at 930 nm and detection at 540 - 700 nm. FLIM measurements were made using a TCSPC module. The laser power was maintained at <80 mW before entering the microscope to avoid photodamage (< 0.3 pW on the sample). The acquisition time was varied in the range of 60-180 seconds per frame depending on the emission intensity from each dye. FLIM data was recorded as a z-stack at different distances from the bottom (z = 0 pm) to the top of the wax layer (ca. 12-20 pm, depending on wax thickness) with a step size of 3-5 pm. FLIM data was analyzed in SPCImage v.8.3 software (Becker & Hickl, Germany) using a model of incomplete exponential decay with offset values fixed to zero. Maximum Likelihood Entropy (MLE) fitting algorithm was used, particularly suitable for low-intensity signals. The minimal binning of pixels (bin 1 , 3x3 pixels) was used for all data to produce a typical peak count in the decay maximum in the range of 100- 200 counts per pixel (which is deemed sufficient for the MLE analysis); thresholding was adjusted from sample to sample to remove pixels of low intensity from the analysis. All decays were fitted using an exponential decay function. A pseudocolor scale was assigned to each fluorescence lifetime, amplitude and the goodness of fit to provide corresponding maps. The lifetime values and errors presented are mean values and standard deviations calculated from histograms corresponding to one FLIM data frame (total amount of pixels in the range of 40 000 - 65536). The reproducibility of data was confirmed in at least two independent experiments; at least three frames were collected per condition.

[0251] Example 2: Ratiometric analysis of local polarity by Nile Red

[0252] The solvatochromic properties of Nile Red were used to track the migration of formulation components into the wax layer (schematically shown in Figure 4). Furthermore, the fluorescence intensity ratio as measured for local polarity changes in the wax film was shown. Figure 5 schematically shows the fluorescence spectrum shift of Nile Red upon polarity changes and indicates the wavelengths ranges suitable for the two channels that are monitored and used for calculating the ratio (channel short and channel long). Figure 6 shows the changes of fluorescence ratios over time upon exposure of the wax film to an aqueous solution of 1000 ppm Plurafac® LF431 at different depth.

[0253] Example 3: Nile red as indicator for penetration of adjuvant and different model actives Adjuvant: Plurafac® LF 1300

[0254] Active-like, fluorescent model ingredients with different LogP values

[0255] NBD-Pz (LogP=0.5) Coumarin 343 (LogP=3.2) FJuorol Yellow088 (LogP=6.4)

[0256] NBZ-Pz = 4-Nitro-7-piperazino-2,1 ,3-benzoxadiazole

[0257] Coumarin 343 = 2,3,6,7-Tetrahydro-11-oxo-1 H,5H,11 H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carboxylic acid

[0258] Fluorol Yellow088 = 2,8-Dimethylnaphtho[3,2,1-kl]xanthene

[0259] Preparation of wax layer

[0260] 500 ppm Nile Red were dissolved in a carnauba wax solution (10 wt.-% wax in CHCh at 80 °C). For preparation of a 120 pm wax layer the solution was coated onto a glass slide. Afterwards the wax layer was heated up to 120 °C for 2 h to release residual chloroform from the layer. CIPAC-water (19.2 °dH) was prepared from a stock solution (1 :100). Formulations comprising the adjuvant Plurafac® LF1300 at a concentration of 1000 ppm and also one of the model-like active ingredient (coumarin 343, Fluorol yellow or NBD-Pz) at a concentration of 500 ppm were prepared with the hard water.

[0261] Ex-situ experiment 1 h: 100 pL of the formulation was applied on the top of the wax layer. After 1 h the droplet was removed by pipette and the wax layer was dried by airflow. The layers were subsequently imaged with the respective contrast.

[0262] Ex-situ experiment 22 h: 100 pL of the formulation was applied on the top of the wax layer, and the wax layer with the droplet of formulation was stored overnight. The layers were subsequently imaged with the respective contrast.

[0263] The results are shown in Figures 7 - 9, wherein A shows one section of a xzy-scan for Nile Red fluorescence at 561 nm, B shows the model active, NBD-Pz, fluorescence at 488 nm, and C shows the maximum projection of a xzy-scan for NBD-Pz fluorescence.

[0264] After 1 h of exposure time, a clear penetration of the active ingredient NBD-Pz in presence of the adjuvant into the wax was observed (Figure 7), visible by an overlay of the local increase in Nile Red fluorescence by penetration of the adjuvant (Fig. 7A) and the fluorescence signals of the model active (Fig. 7B). After 22 h, the effect intensified. The active ingredient Coumarin 343 could penetrate at individual points of the wax layer after an exposure time of 1 h (Figure 8B). Again, the overlay of the adjuvant induced changes of the fluorescence of Nile Red at individual points of the wax layer (Fig. 8A) and the fluorescence of the model active in the wax layer (Fig. 8B) shows that the adjuvant facilitates the penetration of the active into the wax layer. After 22 h, the effect intensified. The observed effect was greater than that for NBD-Pz.

[0265] After 1 h of exposure time, the active ingredient Fluorol Yellow only slightly penetrated into the wax layer (Figure 9B). Even after 22 h, Fluorol Yellow only penetrated at a few areas into the wax layer and the adjuvant had minor influence on its penetration.

Claims

1. Claims1 . Method for determining the penetration properties of at least one compound into and / or through a plant leaf wax cuticular layer, said method comprising a) providing a model plant wax layer comprising at least one wax and Nile Red (9-(Diethylamino)-5 / 7- benzo[a]phenoxazin-5-one), optionally on a solid support, wherein the Nile Red is optionally homogeneously distributed in the model plant wax layer; b) subsequently contacting the model plant wax layer with a composition comprising the at least one compound; and c) monitoring the penetration of the at least one compound into and / or through the model plant wax layer by monitoring the change in Nile Red fluorescence caused by the at least one compound.

2. The method of claim 1 , wherein the compound is a potential adjuvant for agrochemical formulations, preferably a penetration enhancing adjuvant.

3. The method of claim 1 or 2, wherein the at least one wax of the model plant wax layer is a plant wax, preferably a plant wax having a melting point in the range of 40 to 90 °C and / or a hydrocarbon length of 26 to 35 carbon atoms, wherein the plant wax is optionally carnauba wax, candelilla wax or a combination thereof.

4. The method of any one of claims 1 to 3, wherein the model plant wax layer(1) has a thickness of 1 to 30 pm, preferably 2 to 20 pm, more preferably 3 to <20 pm; and / or(2) comprises 1 to 1000 ppm, preferably 50 to 750 ppm, more preferably 200 to 600 ppm, Nile Red.

5. The method of any one of claims 1 to 4, wherein the model plant wax film is provided on a solid support, the solid support preferably being glass, more preferably a glass microscope slide.

6. The method of any one of claims 1 to 5, wherein the contacting is with the surface of the model plant wax layer opposite to the surface contacting the solid support.

7. The method of any one of claims 1 to 6, wherein the composition comprising the at least one compound is a liquid composition, optionally an aqueous solution or dispersion comprising said at least one compound.

8. The method of any one of claims 1 to 7, wherein the composition comprises the at least one compound and an agrochemical active.

9. The method of any one of claims 1 to 8, wherein contacting is carried out for up to 24 hours, preferably 10 minutes to 22 hours.

10. The method of any one of claims 1 to 9, wherein the penetration of the at least one compound into and / or across the model plant wax layer is monitored using fluorescence microscopy, preferably confocal microscopy.11 . The method of any one of claims 1 to 10, wherein Nile Red fluorescence is determined at an excitation wavelength of about 561 nm and / or an emission wavelength of about 565 to about 750 nm.

12. The method of any one of claims 1 to 11 , wherein the penetration properties of said compound are determined by (1) measuring the change in Nile Red fluorescence intensity, (2) measuring and calculating the Nile Red fluorescence intensity ratio between a first channel in the range of about 565 to about 645 nm and a second channel in the range of about 650 to about 745 nm, and / or (3) Nile Red fluorescence lifetime imaging (FLIM).

13. The method of any one of claims 1 to 12, wherein the method is a screening method to determine the penetration properties of a multitude of compounds, preferably in a multiplexing format.

14. Use of Nile Red comprised and optionally homogenously distributed in a wax layer for determining the penetration properties of at least one compound into and / or through said wax layer by monitoring the change in Nile Red fluorescence upon contacting the at least one compound with the wax layer.

15. Wax layer deposited onto a solid support, wherein said wax layer comprises at least one plant wax and Nile Red, wherein the Nile Red is homogeneously distributed in the wax layer, and wherein said solid support is preferably a glass slide.

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