# Patent

## Title
Bioactive acid agrichemical compositions and use thereof

## Abstract

Bioactive agrichemical concentrates and compositions having improved bioactivity comprising metal-acid solutions.

## Background
Provisional Patent Application No. 60/930,913, filed May 18, 2007 and entitled “Bioactive Compositions and Use Thereof” which is hereby incorporated herein in its entirety by reference.


FIELD OF THE INVENTION
The present invention relates to novel bioactive concentrates as liquid solutions or solid compositions comprising an acid and one or more sources of antimicrobial metal ions, alone or, preferably, in further combination with one or more surfactants capable of interacting with cell wall membranes of microorganisms, especially pathogenic microbes. These bioactive concentrates may be diluted with liquid or solid diluents for use in a wide variety of end-use applications, including, in particular, as bioactive agrichemicals for the control, inhibition and/or killing of microorganisms, especially fungi, bacteria and/or plant, stramenophile and fungi-like protists.
BACKGROUND OF THE INVENTION
Bioactive materials for killing or inhibiting the growth and/or proliferation/spreading of bacteria, fungi, and other microorganisms have long been sought and employed in society. Their use dates back centuries, if not thousands of years. Early applications had ranged from pharmaceutical or health related applications to disinfectant and purification applications and more. More recent applications include a whole host of uses, with the largest use, by volume, seen in the agricultural industry. Perhaps one of the earliest bioactive materials was metallic silver and, subsequently, silver salts.
While early bioactive agents were most often metals and simple metal salts, modern science and chemical synthesis have enabled the development and production of synthetic agents, most often organic and organometallic agents, for antibacterial, antifungal and other like applications. Indeed, for many applications, especially pharmaceutical applications, the organic agents have, for the most part, eclipsed the use of inorganic bioactive agents. While inorganic and organometallic materials still command a significant market share of the agrichemical business, their use is limited due to their health and safety concerns, especially from an environment perspective. Indeed, organic bioactive agents command a huge portion of the agrochemical business.
Despite the great success and huge market share/volume commanded by organic pharmaceutical, antibacterial and agrochemical agents, they have not come without cost and consequences. In all areas of applications, a marked and growing trend has emerged: namely the manifestation and spreading of a resistance to such organic agents in most all, if not all, microorganisms. While this resistance is neither universal nor complete, it is growing and involves more and more organic agents. As their resistance grows, so too does their apparent virulence. In this respect, we are all well aware of the growing resistance of bacteria, especially pathogenic bacteria, to traditional pharmaceutical antibiotic agents and the subsequent appearance of what are commonly referred to as superbugs: pathogenic bacteria that show strong resistance to traditional organic antibacterial and pharmaceutical agents.
And, whether a direct or indirect consequence of the appearance of superbugs and/or the growing awareness of the ease by which bacteria can spread combined with an increasing concern for potentially pandemic diseases such as SARS and Bird Flu, we have become a population that is more and more pre-occupied with hygiene and general cleanliness. Consequently, there has been a huge proliferation and exponential growth in the widespread and indiscriminate use and application of cleansers and disinfectants that contain organic antimicrobial agents, all in an effort to ward off exposure to bacteria and, especially, superbugs. However, this indiscriminate use of organic agents has come with, or at least presents the possibility for, an overall increase in antimicrobial resistant organisms. By eradicating the weaker organisms, the stronger and, most often more damaging, organisms are left.
A similar consequence has manifested itself in the agricultural industry as well, especially in that portion relating to crop/food production. The widespread and repetitive use of organic biocides, fungicides, antibacterial agents and the like, has led to the manifestation of less and less efficacy of the same against the targeted diseases: an indicator of a growing resistance. Perhaps more alarming is the speed with which such resistance has begun to appear. For example, despite the great fanfare and promise behind the introduction of strobilurin fungicides in the mid-1990s, resistance had been found after just a couple years use in certain applications. Such a growing trend bodes ill for an industry where fewer and fewer acres are called upon to produce more and more crops to feed the ever growing populations while those organisms and microorganisms responsible for attacking such crops become stronger and stronger and more and more resistant to traditional control means.
While resistance is certainly of great concern, perhaps and even greater concern is the human and environmental toll associated with the widespread use of organic antimicrobial agents. For more than half a century now, more and more scientific literature has appeared correlating long-term exposure (direct and indirect) and use of such organic agrichemicals to various diseases and teratogenic, mutanogenic, and other adverse health consequences in animals and, more importantly, the human population. Perhaps the watershed of this awareness is represented by the outcry relating to the use of DDT and like pesticide agents in the 1960s. In humans, such a correlation of birth defects, cancer, and other diseases with organic agrichemicals is especially disconcerting for those whose water supplies have or may become contaminated with such organic agents due to their and/or their by-product's solubility and long half-lives. Of course drinking water is only one exposure source: another exposure source concern is inhalation from dust blown up from the fields, from wayward aerosols and/or particulates during aerial spraying and dusting, respectively, and from exposure to the clothing of workers who, themselves, were exposed in the fields or during application.
In an effort to move away form organic agents, more recent attention has once again focused on the inorganic agents, including organometallic agents, since these tend to show or have no, or certainly less, tendency to result in resistant bacteria, fungi and the like. However, such a trend merely reawakens the debates and concerns relative to the large-scale dumping of heavy metals in the environment. Although some efforts have recently focused on improving the old, traditional inorganic agents, much more effort, particularly in the non-agricultural arena, has seemingly focused on more complex species and systems, in essence, synthesized inorganic biocides such as the antimicrobial ion-exchange type antimicrobial agents based on zeolites, hydroxy apatite, and zirconium phosphates. Other recent biocides include those based on electrolytically generated silver citrates; thiol-free, specialty complexes of antimicrobial metals; sulfuric acid/sulfate complexes prepared under high pressure and temperature; and the like. While effective, these have limited applications and entail added costs owing to the complex and/or lengthy synthetic processes by which they are prepared. The latter is of especial concern in the agricultural industry where the relative cost to performance trade-off oftentimes precludes the use of functionally very viable options. Here, a few cents per acre difference, even a fraction of a cent difference per acre, can mean the world of difference in the acceptability and utility of a given agent.
Despite their inherent environmental and health concerns, the use of “natural” inorganic agents, including manufactured/processed inorganic agents, has been pushed more and more by various environmental and conservation groups, as well as health advocates, as a favorable replacement to organic agents. While such pressure alone is not likely to change the industry, the growing resistance to organic agents combined with the higher and higher costs of synthetic organic agents, is certainly having an impact: not only in the agricultural industry but across the board in all applications for such bioactive agents. However, as noted above, the reintroduction of and/or increased use of inorganic agents merely brings to the forefront the very issues that caused them to be pushed back to begin with, namely environmental toxicity and contamination and bioaccumulation. Concern is not just for the effects during application, but more so for the long term effects associated with the continual build-up of these inorganic agents or their derivatives, especially the metals, in the environment and in living organisms. Such build-up not only pertains to the soils that are treated but also underground water supplies that may be replenished from the treated fields. Concern also exists, sometimes more so, for the consequences of rain-water run-off carrying the metals into local streams and, again, downstream water supplies. Ultimately, this build-up also occurs in the food chain, with higher and higher concentrations being found in those species in the higher order of the food chain. Ultimately, this affects the human supply chain as seen, for example with mercury and other metals in tuna and swordfish. While many metals, at least at low exposure levels, have little or no affect on humans, their impact is far greater on marine and other aquatic life, especially fish, which tend to be extremely sensitive to heavy metals, like silver, resulting in increase stress and, in extreme exposure situations, widespread kills.
Consequently, as part of this resurgence of inorganic bioactive agents, there has been a significant increase in research and development dollars and time spent to address concerns relative to the use of such inorganic agents. A prime focus has been with respect to making more concentrated materials that, it is hoped, will enable the use of less overall materials. In the agrichemical arena, one of the inorganic agents receiving the greatest attention, owing to its high efficacy, is copper. Indeed, it is thought that copper could see a manifold increase in use as organic agents are either precluded from use or farmers opt out for more natural agents. According to NCFAP data, in 1997 more than 13.7 million pounds of copper was used in fungicide applications as compared to 40 million pounds of synthetic fungicides at a treatment rate of about one-half that of copper. If all synthetic fungicides were to have been supplanted by copper, it would have resulted in an increase of more than 80 million pounds of copper released into the environment. Now, ten years later, though data is not available, one can only assume the amounts are much higher. Furthermore, this is but one use of copper: copper and copper based bioactive compositions are also used in other areas such as algaecides, etc. Regardless, it is clear that any significant shift from synthetic to copper fungicides, algaecides, etc. means a huge impact and release of copper into the environment.
As noted above, recent R&D efforts with inorganic fungicides have focused on the development of improved inorganic agents that produce better effects with less application. Indeed, in August 2006, DuPont, one of the leading manufacturers of agrichemicals, especially copper based fungicides, announced certain breakthroughs, as they described them, in copper fungicides, specifically its Kocide 3000 copper hydroxide based fungicide, touting their ability to provide more antifungal action with less copper. Still, its typical application rate is on the order of 1650 grams of copper per acre per application, with somewhat lower rates, 330 grams per acre, allowed for certain applications. While certainly an improvement over conventional or traditional copper based fungicides which are applied at nearly 4.5 pounds per acre, it still means the intentional release of huge amounts of copper into the environment, even more if the environmentalists are successful in removing or banning the use of more and more organic agents.
Thus, there is still a tremendous need for cost effective, inorganic agrichemical agents that provide good antimicrobial, antifungal, antibacterial, etc., activity without concern for resistance build-up.
In following, there is a need for inorganic antimicrobial, antifungal, antibacterial, etc., agents that may be used universally, or nearly so, without concern, or certainly with reduced concern for environmental contamination and toxicity, especially less so than exists with current inorganic agents.
Similarly, there is a need for inorganic agents that are stable and easy to use, and provide good short term and, preferably, longer term efficacy as compared to many of the current short lived organic agents.
Additionally, there is a need for such inorganic agents that can safely be use in agricultural and horticultural applications, including soil and seed treatment, crop/food producing plants and trees, ornamental and flowing plants and tress, fee and ornamental grasses, and the like with minimal exposure concerns.
Furthermore, there is a need for inorganic bioactive agents that can be use in combination with conventional inorganic and, preferably, organic agrichemical actives and compositions, especially, antimicrobial, antifungal, antibacterial, anti-protist, etc. agents with synergistic results; thus, enabling less use, overall, of such actives.
Finally, there is a need for bioactive agents that provide efficacious antimicrobial, antifungal, antiprotist, and/or antibacterial performance with minimal release of inorganic metals to the environment.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there are provided solid form bioactive concentrates comprising an acid, especially a weak or moderate acid, at least one source of at least one antimicrobial metal ion, and, optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, said acid being present in at least 40 weight percent, preferably from 40 to 80 percent, based on the total weight of the bioactive acid composition, and at a level that represents at least a 2 times molar excess relative to the antimicrobial metal ions of the source, said bioactive acid composition having a pH of less than 6, preferably from about 1.5 to 5, when diluted in a solvent, especially water, to a point where the amount of antimicrobial metal ion is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions. These solid concentrates may be let down by dry blending with solid diluents or fillers or they may be diluted and transformed into liquid form using liquid diluents or fillers for use in various end-use applications.
According to a second aspect of the present invention there are provided liquid form bioactive concentrates comprising a concentrated aqueous or aqueous-based acid solution, especially of a weak or moderate acid, at least one antimicrobial metal ion or antimicrobial metal ion source, fully or partially dissolved in said acid solution, and, optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, wherein the concentration of the acid, based on the total weight to the bioactive acid solution is at least about 40 weight percent, preferably from 40 to 80 percent, acid and the acid is present a level that is at least a 2 times molar excess relative to the antimicrobial metal ion(s), and wherein, the pH of the bioactive acid solution is less than 6, preferably 1.5 to 5, when the concentrated bioactive acid solution is diluted in a solvent, especially water, to a point where the amount of antimicrobial metal ion is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions. These liquid concentrates may be let down by mixing with an appropriate solvent, most especially water or an aqueous-based solvent, for application. Alternatively, especially for agricultural, including horticultural, applications, the concentrate may be applied as is or in a diluted state to a solid, absorbent material for ultimate end-use application.
According to a third embodiment of the present invention, there are provided flowable bioactive agrichemical compositions in particle form, e.g., dusts, granules, powders, or combinations thereof, comprising a solid carrier particle which has been treated with a bioactive acid solution, especially an aqueous or aqueous-based solution, having a pH of less than 6, preferably 1.5 to 5, and comprising an acid, especially a weak or moderate acid, at least one antimicrobial metal ion source, and, optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, said acid present in a molar excess, relative to the antimicrobial metal ions, where the amount of antimicrobial metal ion is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions.
According to a fourth embodiment of the present invention, there are provided flowable bioactive agrichemical compositions in particle form, e.g., dusts, granules, powders, or combinations thereof, comprising a bioactive acid composition in particle form, preferably a powder, said bioactive agrichemical composition having been made by i) forming an aqueous solution of an acid, especially a weak or moderate acid, at least one antimicrobial metal ion source, and, optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, said acid present in a molar excess, relative to the antimicrobial metal ions, where the amount of antimicrobial metal ion is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions, ii) allowing the water to evaporate to form the solid bioactive acid and iii) if necessary, grinding or crushing the formed solid bioactive acid to form a powder.
According to a fifth embodiment of the present invention there are provided liquid bioactive agrichemical compositions comprising a) a suitable solvent or solvent system, b) an acid, especially a weak or moderate acid, c) at least one antimicrobial metal ion source, and d) optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, wherein the solution of the acid and solvent has a pH of less than 6, preferably 1.5 to 5, prior to, and preferably after as well, the addition of the conventional bioactive agrichemical active, said acid (b) is present in a molar excess relative to the antimicrobial metal ions of the source (c) and wherein the amount of antimicrobial metal ions attributed to the source (c) is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions. Most preferably, the solvent is water or a water-based solvent system, most preferably water, and the acid and the antimicrobial metal ion source are all wholly or substantially soluble or miscible in the solvent. Where desired, it is also possible to add the solution to a second, non-aqueous solvent, including a lipophilic solvent, to form a suspension or emulsion.
According to a sixth embodiment of the present invention there is provided a method of making a solid form bioactive agrichemical concentrate comprising dry mixing a) an acid, especially a weak or moderate acid, b) at least one source of at least one antimicrobial metal ion, and c) optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, said acid being present in at least 40 weight percent, based on the total weight of the acid and antimicrobial metal ions source, and at a level that represents at least a 2 times molar excess relative to the antimicrobial metal ions of the source, the acid being present in an amount whereby a solution made by adding the acid and the antimicrobial metal ions source to water will have a pH of less than 6, preferably from about 1.5 to 5, when diluted to a point where the amount of antimicrobial metal ion is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions.
According to a seventh embodiment of the present invention there is provided a method of making a solid form bioactive concentrate comprising i) dissolving a) an acid, especially a weak or moderate acid, b) at least one source of at least one antimicrobial metal ion, and c) optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, in a volatile solvent, especially water or a water-based solvent, ii) allowing the solvent to evaporate to leave a solid cake, granules, or powder, and iii) if necessary or desired, grinding the cake to form granules or a powder, wherein the acid is present in at least 40 weight percent, based on the total weight of the acid and antimicrobial metal ions source, and at a level that represents at least a 2 times molar excess relative to the antimicrobial metal ions of the source, the acid being present in an amount whereby a solution made by adding the acid and the antimicrobial metal ions source to water will have a pH of less than 6, preferably from about 1.5 to 5, when diluted to a point where the amount of antimicrobial metal ion is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions.
According to an eighth embodiment of the present invention there is provided a method of making a liquid bioactive agrichemical concentrate said method comprising forming a concentrated aqueous or aqueous-based acid solution of an acid, preferably a weak or medium acid, wherein the concentration of the acid is at least 40% by weight, dissolving in the concentrated acid solution at least one antimicrobial metal ion source and, optionally, though preferably, at least one water-soluble surfactant, preferably an anionic, non-ionic and/or amphoteric surfactant.
According to a ninth embodiment of the present invention there is provided a method of preventing or inhibiting the growth of plant pathogens, especially fungi, bacteria and/or plant, stramenophile and fungi-like protists in agricultural, including horticultural, applications, said method comprising applying to the seeds of the pertinent crop or plant; to the soil in which the seed, crop or plant is or is to be planted; to the aqueous environment in which the plants are growing; or to the matter of the plant itself, a flowable bioactive agrichemical composition in particle form comprising a solid carrier particle which has been treated with a bioactive acid solution, especially an aqueous or aqueous-based solution, having a pH of less than 6, preferably 1.5 to 5, and comprising an acid, especially a weak or moderate acid, at least one antimicrobial metal ion source, and, optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, said acid present in a molar excess, relative to the antimicrobial metal ions, where the amount of antimicrobial metal ion is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions. Most preferably, the composition according to this method will further comprise at least one agent for adhering or enhancing the adherence of the bioactive agrichemical actives to the matter being treated. Generally speaking, the inventive bioactive agrichemical composition will be applied at a rate or amount whereby the amount of the bioactive acid solution originated antimicrobial metal ion(s) applied will be no more than about 500 grams per acre, preferably no more than 250 grams per acre.
According to a tenth embodiment of the present invention there is provided a method of preventing or inhibiting the growth of plant pathogens, especially fungi, bacteria and/or plant, stramenophile and fungi-like protists in agricultural, including horticultural, applications, said method comprising applying to the seeds of the pertinent crop or plant; to the soil in which the seed, crop or plant is or is to be planted; to the aqueous environment in which the plants are growing; or to the matter of the plant itself, a flowable bioactive agrichemical composition in particle form said bioactive agrichemical composition having been made by: i) forming an aqueous solution of an acid, especially a weak or moderate acid, at least one antimicrobial metal ion source, and, optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, said acid present in a molar excess, relative to the antimicrobial metal ions, where the amount of antimicrobial metal ion is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions, ii) allowing the water to evaporate to form the solid bioactive acid and iii) if necessary, grinding or crushing the formed solid bioactive acid to form a powder. Most preferably, the composition according to this method will further comprise at least one agent for adhering or enhancing the adherence of the bioactive agrichemical actives to the matter being treated. As with the previous embodiment, the formulation of the inventive bioactive agrichemical composition will be such that the antimicrobial metal ion(s) applied will be no more than about 500 grams per acre, preferably no more than 250 grams per acre.
According to an eleventh embodiment of the present invention there is provided a method of preventing or inhibiting the growth of plant pathogens, especially fungi, bacteria and/or plant, stramenophile and fungi-like protists in agricultural, including horticultural, applications, said method comprising applying to the seeds of the pertinent crop or plant; to the soil in which the seed, crop or plant is or is to be planted; to the aqueous environment in which the plants are growing; or to the matter of the plant itself, liquid bioactive agrichemical compositions comprising a) a suitable solvent or solvent system, b) an acid, especially a weak or moderate acid, c) at least one antimicrobial metal ion source, and d) optionally, though preferably, at least one surfactant, especially at least one anionic, non-ionic and/or amphoteric surfactant, that impacts or interacts with cell wall membranes of microorganisms, especially pathogenic microbes, or the function thereof, wherein the solution of the acid and solvent has a pH of less than 6, preferably 1.5 to 5, prior to, acid present in a molar excess relative to the antimicrobial metal ions of the source (c) and wherein the amount of antimicrobial metal ions attributed to the source (c) is 500 ppm or less in the case of a single ion or 1000 ppm or less in the case or multiple antimicrobial metal ions. Most preferably, the solvent is water or a water-based solvent system, most preferably water, and the acid and the antimicrobial metal ion source are all wholly or substantially soluble or miscible in the solvent. Generally speaking, this formulation of the inventive bioactive agrichemical composition will be such that, when applied in use, the application rate or amount of the bioactive acid solution originated antimicrobial metal ion(s) applied will be no more than about 500 grams per acre, preferably no more than 250 grams per acre.
Inasmuch as a key objective of the present invention is to lessen the amount of metals entering the environment, preferred embodiments of each of the foregoing embodiments will such that the contribution of antimicrobial metal ions from the antimicrobial metal ion source will be no more than 300 ppm, most preferably no more than 50 ppm, in the case of a single antimicrobial metal ion and no more than 500 ppm, preferably no more than 150 ppm, in the case of multiple antimicrobial metal ions.
Inasmuch as it is also an objective of the present invention to avoid the use of materials that induce phytotoxicity, the acids are preferably organic acids, most preferably carboxylic acids, and/or the bioactive acid solution, in the diluted state has a pH of greater than 2 and less than 6.


DETAILED DESCRIPTION
The present invention embraces many different embodiments, as set forth above, all of which have significant degree of common characteristics and make-up. Yet, while the foregoing sets forth each embodiment in its most general respect, there are many aspects of each that are, in certain embodiments, common and others that are unique to their embodiments, In its most fundamental respect, the invention pertains to bioactive concentrates comprising a) one or more antimicrobial metal ions or ion sources, b) an acid or in an acidic solution, and c) optionally, though preferably, one or more surfactants for combating or preventing the growth or proliferation of fungi, bacteria, viruses, and plant , stramenophile and fungi-like protists, as well as bioactive agrichemical products and formulated based on diluted forms of the foregoing. These concentrates and agrichemical products may exist in solid or liquid form and may include any number of additives typical for their intended end-use application. As bioactive agrichemical materials and products, these compositions are especially useful, and most often synergistically, when combined with or made to include a conventional agrichemical active of formulated active. The latter compositions are more fully discussed in and the subject of International Patent Application Publication No. WO 2008/144015 and United States Published Patent Application Nos. U.S. 2008/0292673 A1, U.S. 2008/0292674 A1, and U.S. 2008/0299222 A1, all of which are entitled “Bioactive Agrichemical Compositions and Use Thereof ”and were filed on the same day as this application with the same inventors as the present application, all of which are incorporated herein in their entirety by reference.
For convenience in drafting and simplicity in reading this application, the word “bioactive” is intended to include agents that kill or prevent or inhibit the growth and/or proliferation of bacteria, fungi, viruses, and plant, stramenophile and fungi-like protests, particularly those that affect or adversely affect, even from a purely esthetic standpoint, plants and trees, especially those associated with the agricultural and horticultural products including food crops, feed crops, flowers, ornamentals, turf, and the like, i.e., plant pathogens. Oftentimes, the present invention is discussed in terms of “fungicides” and “antifungal activity” as well as fungicidal actives; though it is to be understood that it is not limited thereto.
Similarly, the word “active” refers to those compounds or compositions that are directly responsible for the bioefficacy of the bioactive agrichemical in addressing or attacking the target organism. A formulated active is one that in addition to the active ingredient also contains one or more other constituents that may or may not influence the bioefficacy of the active, but in any event are not themselves directly responsible for killing or preventing the growth of the targeted microorganism. Further, as a matter of clarification, the terms “bioactive acid composition” and “bioactive acid solution” are oftentimes used herein and relate to the solid and liquid forms, respectively, of the concentrates and diluted forms of the compositions of the present invention, or both are referred to as the “bioactive acid solution or composition.”
The acids that may be used in the present invention are either solid or liquid in their natural state, but are readily dissolved in or miscible with water or an aqueous based solvent or the organic carrier or diluent employed for the particular application to be addressed. For example, if one is intending to make an oil or oil-based (or other non-aqueous solvent based) fungicide, e.g., for use in an aqueous environment, either the components of the bioactive composition must be soluble in or miscible with the chosen oil or other non-aqueous or lipophilic solvent or the aqueous or aqueous-based bioactive acid solution is to be combined with the oil or other non-aqueous or lipophilic solvent to form an emulsion or suspension.
Exemplary acids include the organic acids, especially the carboxylic acids such as citric acid, valeric acid, itaconic acid, acetic, citriconic acid, lactic acid, malic acid, succinic acid, aldaric acid, malonic acid, proprionic acid, malonic acid, maleic acid, salicylic acid, glutaric acid, tartaric acids, benzoic acid and the like, as well as the mineral acids such as nitric acid, sulfuric acid, phosphoric acid, boric acid, and the like. The preference is for weaker or moderate acids such as aldaric, citric, malic, and lactic acids as opposed to the moderate to strong mineral acids like boric and phosphoric acids. However, strong acids, especially strong mineral acids like sulfuric or nitric acid, may be used; however, depending upon the strength of the acid, it may be preferable to buffer the acid so as to avoid handling and use problems, especially problems associated with the substrate to which the bioactive composition is to be applied. This is particularly important for bioactive agrichemical compositions to be applied to plants, animals and crops or foodstuffs since the acid may damage the substrate, directly or indirectly. In plants, for example, there is considerable concern for phytotoxicity resulting from acid treatments alone or in combination with metal compounds such as copper fungicides and the like. Thus, while efficacious, it is most preferable to avoid mineral acids and, instead, employ carboxylic acids. Additionally, though some suitable acids fall outside of this range, it is desirable that the pKa (in water @25° C.) of the acid be greater than 0, preferably greater than 1, most preferably greater than 1.5.
Generally speaking, and despite the strong efficacy of phosphoric acid and nitric acid, it is preferred that weak or moderate acids be used. This is especially desirable as it avoids the potential need for buffering agents, which are not desired. While it is to be appreciated that other surfactants, fungicides, wetting agents, emulsifiers, and the like that may be, and, depending upon the end use application, are likely to be added to the inventive compositions of the present invention, may have a buffering effect on the pH of these bioactive systems, this effect is not an intended or necessarily desirable effect. Indeed, it may be necessary to add more acid to the composition in order to maintain the required level of acidity.
As noted, acidity is critical to the efficacy of the bioactive concentrates and compositions, especially agrichemical compositions, of the present invention. Generally speaking, the pH of the bioactive acid solution or bioactive acid composition prepared using purified water will be less than 6, preferably from about 1.5 to 5 and more preferably from about 2 to about 4, most preferably greater than 2. Most preferably, the fully for lated end-use products, especially the bioactive agrichemical compositions, of the present invention will also meet the foregoing pH limitations when prepared or when diluted with purified water to that concentration that is to be applied. In the case of assessing or confirming the pH of the solid bioactive composition or a solid bioactive agrichemical composition according to the present invention, the bioactive composition or, as appropriate, the bioactive agrichemical is first dissolved in purified water to a concentration equivalent to that at which it would be applied in use, and the pH measured. As acknowledged in the technical art generally, purified water has a neutral pH,  ̃pH 7.
The second critical aspect of the acid concentration relates to the molar equivalence to the antimicrobial metal ions present in the bioactive acid composition or bioactive acid solution. At a minimum, there must be a 2 times molar excess, though preferably there is at least a 5 times, and most preferably at least a 10 times, molar excess acid. These levels are typically attained by formulating bioactive acid solutions whereby the acid concentration in the final diluted state of the bioactive composition is from about 0.01% to about 10%, preferably from about 0.1% to about 4% by weight of the solution. Higher concentrations may also be used, e.g., up to 20% or more, provided that the substrate to which the bioactive composition is to be applied is not affected by the higher acid content and/or the acid is a weak or weakly moderate acid. Certainly, higher concentrations will be used in the production of concentrates as discussed below.
The second critical component of the bioactive compositions is the antimicrobial metal ion: more aptly its metal ion source. Suitable metal ions are selected from the group consisting antimicrobial transition metal ions and poor ions that have shown antimicrobial bioefficacy. Preferred metal ions are selected from the group consisting of silver, copper, zinc, mercury, tin, gold, lead, iron, bismuth, cadmium, chromium and thallium ions or combinations of any two or more of the foregoing. Most preferably, the metal ions are selected from the group consisting of silver, copper and zinc ions and combinations of any two or all three. Bioactive compositions in which at least two and preferably all three of these preferred ions are present are especially beneficial and preferred. Where multiple antimicrobial metal ions are present, each will be present in a molar amount of 3 to 97 percent, preferably 9 to 91 percent, more preferably 20 to 80 percent. In its preferred embodiment, where multiple metal ions are present, they will be present in an equal amount whereby no one metal ion is more than 20 times, more preferably no more than 10 times that of any other metal ion. Especially good results have been found where each antimicrobial metal ion is present in an equal amount, by weight.
The metal ion is added to the acid solution or, as appropriate, the acid, in the form of a source compound, salt or complex that readily releases the ions or otherwise dissociates in the acid solution or when the source and acid are dissolved in a solvent, especially water or a water-based solvent. Exemplary salts and organometallic compounds that may suitably serve as the ion sources include the respective oxides, sulfides, carbonates, nitrates, phosphates, dihydrogen phosphates, sulfates, oxalates, quinolinolates, thiosulfates, sulfonates, phthalates, hydroxides, glycolates, and the like of the antimicrobial metals as well as the carboxylic acid salts thereof, especially the simple carboxylates, such as the citrates, benzoates, acetates, lactates, etc. of said antimicrobial metals. Other salts such as the halide salts and substituted halide salts, such as the halides, hexafluoroantimonates, tetrafluoroborates, and perchlorates of said antimicrobial metals may be used though they are less desirable as they tend to have slow and/or poor solubility, especially in water. Specific metal ion sources include, but are certainly not limited to, silver nitrate, silver oxide, silver acetate, silver citrate, cupric oxide, copper hydroxide, cuprous oxide, copper oxychloride, cupric acetate, copper quinolinolate, copper citrate, zinc oxide, zinc citrate, and the like.
It has also been surprisingly found that certain inorganic complexes may also serve as the metal ion source. Specifically, ion-exchange type antimicrobial agents and dissolving glass antimicrobial agents may be used where the carrier matrix of these materials is soluble in the acid or diluted acid. For example, it has been found that zeolites are readily soluble in concentrated citric acid. Here the metal ion source or sources are added to the acid with mixing until the particles are dissolved. It is also contemplated that these metal ion sources may be only partially dissolved so as to provide for a longer term source of the antimicrobial metal ion. While these ion sources tend to dissolve in the diluted acid, to speed up and/or enhance the dissolving of the metal ion source, it is preferable to dissolve them in a concentrated acid solution, preferably one of from about 40% to 80% concentration.
Suitable ion-exchange type agents include, but are not limited to aluminosilicates, zeolites, hydroxyapatite, and zirconium phosphates, all of which are commercially available and/or fully described in the patent literature. For example, antimicrobial metal ion-containing hydroxyapatite particles are described in, e.g., U.S. Pat. Nos. 5,009,898 and 5,268,174; antimicrobial metal ion-containing zirconium phosphates are described in, e.g., U.S. Pat. Nos. 4,025,608; 4,059,679; 5,296,238; 5,441,717 and 5,405,644 as well as in the Journal of Antibacterial and Antifungal Agents, Vol. 22, No. 10, pp. 595-601, 1994; and antimicrobial metal ion-containing aluminosilicates and zeolites are described in, e.g., U.S. Pat. Nos. 4,911,898; 4,911,899; 4,938,955; 4,938,958; 4,906,464; and 4,775,585, all of the aforementioned patents hereby being incorporated herein by reference in their entirety. Suitable soluble glasses include those described in, e.g., U.S. Pat. No. 5,470,585, which is also incorporated herein by reference in its entirety.
While individual metal ion sources may be used, it is also desirable to use combinations of metal ion sources so as to provide a mixture of metal ions. In certain instances, a single source may provide multiple metal ions. For example, preferred ion-exchange type metal ion sources include AgION AJ10D which contains both silver and zinc ions and AgION AC10D which includes both silver and copper ions. Most preferably, the metal ion sources are the readily soluble salts and compounds, as mentioned above, and most preferably the combination of such compounds whereby solutions having equal or relatively equal concentrations of each of silver, copper and zinc ions are prepared. Suitable combinations include combinations of silver citrate, copper citrate and zinc citrate as well as combinations of silver nitrate, copper sulfate and zinc oxide.
The amount of the antimicrobial metal ion source to be incorporated into the acid solution or, as appropriate, to be combined with the acid is that which is sufficient to provide a concentration of from about 1 ppm to about 500 ppm, preferably from about 1 ppm to about 300 ppm, more preferably about 2 ppm to about 100 ppm, most preferably from about 5 to about 50 ppm of each antimicrobial metal ion, in the bioactive acid solution or bioactive acid composition at its diluted, end-use concentration. Where multiple metal ions and/or metal ion sources are used to provide combinations of metal ions, the total concentration of metal ions in the solutions should be from about 2 ppm to about 1000 ppm, preferably from about 2 ppm to about 500 ppm, more preferably from about 5 ppm to 300 ppm, most preferably from about 5 ppm to about 150 ppm, in the bioactive acid solution or bioactive acid composition at its diluted, end-use concentration. Of course higher levels could be used but are not necessary to provide suitable bioefficacy and, more importantly, such higher use conflicts with the desired intent of minimizing metal addition to the environment. Thus, in following with said objective, it is preferable to use the minimal, or nearly so, amount possible for the desired application.
In agricultural and horticultural applications, phytotoxicity is especially of concern. Thus, in accordance with the agricultural and horticultural applications of this invention, especially for application to seedlings and plants, the level of the metals should be less than would otherwise cause phytotoxicity. Most preferably, as noted above, the objective is to use as low a level of metal ion as is reasonably possible yet continue to provide the benefits desired, especially fungicidal, protisticidal, and/or antimicrobial properties. This concern is especially pertinent to those compositions containing copper alone or in combination with one or more of the other antimicrobial metal ions and most especially, where the bioactive acid solution or composition is to contain or be used in conjunction with another copper or copper-based material. In this respect, it should be noted that the aforementioned limitations on the antimicrobial metal ions refers only to those antimicrobial metal ions contributed by the one or more sources of antimicrobial metal ions associated with the bioactive acid solution or bioactive acid composition, and not to the copper or any other antimicrobial metals or metal ions that may be contributed by other compounds or materials to be used in conjunction or in combination with the bioactive acid solutions or bioactive acid compositions.
Optionally, though preferably, the bioactive acid solutions or bioactive acid compositions, and, in any event, the bioactive agrichemical compositions of the present invention include one or more surfactants, especially water soluble surfactants. Although good results have been achieved in weak and moderate acid bioactive acid solutions without the surfactants, the use of the surfactant should be and is generally preferred with such acids. Furthermore, while certain strong and very strong acids, especially mineral acids, do not warrant the need for surfactants, e.g., phosphoric acid, it is especially desirable, and in some instances necessary, e.g., where other than only short term bioefficacy is desired, to employ one or more surfactants. Especially preferred surfactants are those that affect or interact with cell walls or membranes of microorganisms, especially pathogenic microbes, or their function. Suitable surfactants include anionic, cationic, non-ionic and amphoteric (e.g., zwitterionic) surfactants, especially those that are water soluble or show relatively good water solubility. Preferably the surfactants are anionic, non-ionic and/or amphoteric surfactants such as the sulfonates, sulfates, sulfosuccinates, sarcosinates, mono and diglycerides, amine oxides, ether carboxylates, betaines, sulfobetaines, gylcinates and the like. Generally, cationic and those non-ionic surfactants having polyalkylether units, especially polyethylene oxide units, with degrees of polymerization of the alkylene ether unit of greater than about 6 do not show the same level of effectives in providing synergy to the bioactive compositions as the other surfactants. Nonetheless, such surfactants may be used in combination with effective surfactants so long as they do not materially detract from or reduce the bioefficacy of the compositions.
The surfactant is typically used in conventional amounts, i.e., will be added to the bioactive acid solutions or bioactive acid compositions in an amount whereby the concentration of the surfactant in the end-use diluted state of the bioactive agrichemical compositions is consistent their use level in traditional fungicides. Generally speaking, the surfactant will be present in an amount of from about 0.001% to about 3%, preferably from about 0.01% to about 0.5%, by weight based on the total weight of the bioactive acid solution or bioactive acid composition in the diluted state. While higher loadings could be used, it is not necessary to manifest the desired synergy in bioefficacy. Generally, where the surfactant is basic in nature or one that hydrolyzes in water to form a basic solution, the amount should be minimized and/or the amount of acid increased so as to avoid too much neutralization of the bioactive acid solution.
Exemplary anionic surfactants and classes of anionic surfactants suitable for use in the practice of the present invention include: alcohol sulfates; alcohol ether sulfates; alkylaryl ether sulfates; alkylaryl sulfonates such as alkylbenzene sulfonates and alkylnaphthalene sulfonates and salts thereof; alkyl sulfonates; mono- or di-phosphate esters of polyalkoxylated alkyl alcohols or alkylphenols; mono- or di-sulfosuccinate esters of C12 to C15 alkanols or polyalkoxylated C12 to C15 alkanols; alcohol ether carboxylates; phenolic ether carboxylates; polybasic acid esters of ethoxylated polyoxyalkylene glycols consisting of oxybutylene or the residue of tetrahydrofuran; sulfoalkylamides and salts thereof such as N-methyl-N-oleoyltaurate Na salt; polyoxyalkylene alkylphenol carboxylates; polyoxyalkylene alcohol carboxylates alkyl polyglycoside/alkenyl succinic anhydride condensation products; alkyl ester sulfates; naphthalene sulfonates; naphthalene formaldehyde condensates; alkyl sulfonamides; sulfonated aliphatic polyesters; sulfate esters of styrylphenyl alkoxylates; and sulfonate esters of styrylphenyl alkoxylates and their corresponding sodium, potassium, calcium, magnesium, zinc, ammonium, alkylammonium, diethanolammonium, or triethanolammonium salts; salts of ligninsulfonic acid such as the sodium, potassium, magnesium, calcium or ammonium salt; polyarylphenol polyalkoxyether sulfates and polyarylphenol polyalkoxyether phosphates; and sulfated alkyl phenol ethoxylates and phosphated alkyl phenol ethoxylates; sodium lauryl sulfate; sodium laureth sulfate; ammonium lauryl sulfate; ammonium laureth sulfate; sodium methyl cocoyl taurate; sodium lauroyl sarcosinate; sodium cocoyl sarcosinate; potassium coco hydrolyzed collagen; TEA (triethanolamine) lauryl sulfate; TEA (Triethanolamine) laureth sulfate; lauryl or cocoyl sarcosine; disodium oleamide sulfosuccinate; disodium laureth sulfosuccinate; disodium dioctyl sulfosuccinate; N-methyl-N-oleoyltaurate Na salt; tristyrylphenol sulphate; ethoxylated lignin sulfonate; ethoxylated nonylphenol phosphate ester; calcium alkylbenzene sulfonate; ethoxylated tridecylalcohol phosphate ester; dialkyl sulfosuccinates; perfluoro (C6-C18)alkyl phosphonic acids; perfluoro(C6-C18)alkyl-phosphinic acids; perfluoro(C3-C20)alkyl esters of carboxylic acids; alkenyl succinic acid diglucamides; alkenyl succinic acid alkoxylates; sodium dialkyl sulfosuccinates; and alkenyl succinic acid alkylpolyglykosides.
Exemplary amphoteric and cationic surfactants include alkylpolyglycosides; betaines; sulfobetaines; glycinates; alkanol amides of C8 to C18 fatty acids and C8 to C18 fatty amine polyalkoxylates; C10 to C18 alkyldimethylbenzylammonium chlorides; coconut alkyldimethylaminoacetic acids; phosphate esters of C8 to C18 fatty amine polyalkoxylates; alkylpolyglycosides (APG) obtainable from a acid-catalyzed Fischer reaction of starch or glucose syrups with fatty alcohols, in particular C8 to C18 alcohols, especially the C8 to C10 and C12 to C14 alkylpolyglycosides having a degree of polymerization of 1.3 to 1.6., in particular 1.4 or 1.5.
Exemplary non-ionic surfactants and classes of non-ionic surfactants include: polyarylphenol polyethoxy ethers; polyalkylphenol polyethoxy ethers; polyglycol ether derivatives of saturated fatty acids; polyglycol ether derivatives of unsaturated fatty acids; polyglycol ether derivatives of aliphatic alcohols; polyglycol ether derivatives of cycloaliphatic alcohols; fatty acid esters of polyoxyethylene sorbitan; alkoxylated vegetable oils; alkoxylated acetylenic diols; polyalkoxylated alkylphenols; fatty acid alkoxylates; sorbitan alkoxylates; sorbitol esters; C8 to C22 alkyl or alkenyl polyglycosides; polyalkoxy styrylaryl ethers; alkylamine oxides; block copolymer ethers; polyalkoxylated fatty glyceride; polyalkylene glycol ethers; linear aliphatic or aromatic polyesters; organo silicones; polyaryl phenols; sorbitol ester alkoxylates; and mono- and diesters of ethylene glycol and mixtures thereto; ethoxylated tristyrylphenol; ethoxylated fatty alcohol; ethoxylated lauryl alcohol; ethoxylated castor oil; and ethoxylated nonylphenol; alkoxylated alcohols, amines or acids, mixtures thereof as well as mixtures thereof with diluents and solid carriers, in particular clathrates thereof with urea. The alkoxylated alcohols, amines or acids are preferably based on alkoxy units having 2 carbon atoms, thus being a mixed ethoxylate, or 2 and 3 carbon atoms, thus being a mixed ethoxylate/propoxylated, and having at least 5 alkoxy moieties, suitably from 5 to 25 alkoxy moieties, preferably 5 to 20, in particular 5 to 15, in the alkoxy chain. The aliphatic moieties of the amine or acid alkoxylated may be straight chained or branched of 9 to 24, preferably 12 to 20, carbon atoms. The alcohol moiety of the alcohol alkoxylates is as a rule derived from a C9-C18 aliphatic alcohol, which may be non-branched or branched, especially monobranched. Preferred alcohols are typically 50% by weight straight-chained and 50% by weight branched alcohols.
As noted above, the aforementioned surfactants may be used alone or in combination. Furthermore, while not all of the surfactants mentioned above will provide the desired synergy when used alone with the metals and acids, depending upon the ultimate end-use application for the bioactive agrichemical compositions of the present invention, they may nevertheless be used in combination with the synergistic surfactants for their intended function. For example, certain of the aforementioned surfactants may enhance the dispersion of the actives in the solvent or may enhance the wetting out of the substrate to which the inventive bioactive compositions of the present invention are applied, e.g., plants, seeds, and soil in the case of agrichemical compositions and countertops, touch surfaces, etc. in the case of disinfectants. All of these surfactant materials are well known and commercially available. Furthermore, those skilled in the art, without undue experimentation, will readily appreciate which surfactants and/or combinations of surfactants, in addition to the synergist surfactants, may be used for the specific end-use application. Again, it is important that when additional surfactants are employed for other purposes they not interfere with or have minimal interference with the synergy that results from the desired surfactants, i.e., those that show synergy in providing antimicrobial, including antibacterial and/or antifungal, activity when used in combination with the acid and metal ions.
If any interference exists and the other surfactant is necessary or otherwise desired for the application, then its use should be minimized to produce the least adverse impact on the synergy and/or attributes of the active components of the inventive bioactive agrichemicals of the present invention while manifesting the desired property for which it is to be employed. Furthermore, if there is concern with such interference, especially if the surfactants are used or to be used in an amount that will neutralize the acid of the bioactive compositions so as to render them outside of the claimed range, then those surfactants may still be added but not until the time of application. In essence the bioactive compositions of the present inventions may be employed as two- or more part systems to be mixed when applied or when preparing the diluted compositions, which are then to be immediately applied. Most preferably, it is best to avoid the use of such surfactants or those amounts of said surfactants that will adversely affect the bioefficacy of the claimed compositions.
As noted above, the bioactive compositions of the present invention may be used in conjunction or in combination with one or more other conventional bioactive agrichemical actives or formulations suitable for the intended end-use. Furthermore, as also noted above and in the aforementioned co-pending, co-filed pending International and United States patent applications, the combined use of the claimed bioactive acid solution or composition with other conventional bioactive agrichemical actives and formulations, particularly fungicides, oftentimes results in enhanced bioefficacy, a synergy that is otherwise unexpected. For example, previously non-efficacious levels of conventional bioactive actives are rendered efficacious as a result of the presence of the bioactive acid solution or composition. Similarly, these combinations oftentimes enable one to achieve the same level of bioefficacy with less than conventional application rates or amounts of the conventional bioactive agrichemical active. Additionally, and of particular significance, the combination is also believed to reduce the incidence of and/or the speed with which bio-resistance to conventional agrichemicals, especially the synthetic organic agrichemicals, is manifested in target organisms. Thus, the commercial life expectancy of these and future conventional agrichemical actives is likely to be increased and the generation of superbugs or resistant strains of the bacterial fungi, protists and the like decreased or delayed.
The bioactive agrichemical compositions according to the present invention can be used alone or, preferably and advantageously, they are used in combination with (typically as a mixture) one or more other compatible components or additives typical of agrichemical treatments and compositions including, for example, solid or liquid fillers or diluents, adjuvants, surfactants or equivalents, which are suitable for the desired use and which are acceptable for use, from an environmental, health and safety as well as regulatory perspective, in the particular intended end-use application. This is especially so for those applications where the bioactive compositions are to be used in agriculture: whether as a soil, seed, or plant treatment or in the treatment of foodstuffs, prior to or following harvest. In following, the formulations can also contain ingredients of other types, such as protective colloids, adjuvants, binders, rain fasteners, thickeners, thixotropic agents, penetrating agents, oils for spraying, stabilizers, antifreeze agents, defoaming agents, foaming agents, corrosion inhibitors, dyes, or the like, as well as other known active ingredients which have pesticidal properties (in particular fungicidal, insecticidal, acaricidal or nematicidal properties) or, in the case of in-field agricultural applications, which have plant-growth-regulating properties.
The nature and amount of the additives to be employed in the bioactive agrichemical compositions of the present invention depends, in part, upon the end-use application and the form in which the composition is to take. Specifically, the bioactive compositions of the present invention may be in the form of and/or manufactured into e.g. emulsion concentrates, solutions, oil in water emulsions, wettable powders, soluble powders, suspension concentrates, dusts, granules, water dispersible granules, micro-capsules, gels, tablets and other formulation types by well-established procedures. The specific procedure typically includes intensive mixing and/or milling of the bioactive compositions with the other substances. The form of application such as spraying, atomizing, dispersing, dusting, pouring and the like may be chosen based on the compositions to be applied, the desired objectives, and the given circumstances.
Although the typical definition of “filler” is a material added for the primary purpose of adding bulk, in the present application, “fillers” typically have function and utility and generally refer to organic or inorganic, natural or synthetic components with which the active components are combined to facilitate their application, for example, onto plants, seeds or the soil, for example as a carrier. These fillers are generally inert and must be acceptable for the intended application, especially for agronomic uses, in particular for treating plants.
The filler can be solid, for example clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (for example ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, calcium carbonate, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earths, or synthetic minerals, such as silica, alumina or silicates, in particular aluminium or magnesium silicates. The solid fillers which are suitable for granules are as follows: natural, crushed or broken rocks, such as calcites, marble, pumice, sepiolite or dolomite; synthetic granules of inorganic or organic flours; granules of organic material such as sawdust, coconut shell, corn ear or envelope, or tobacco stem; kieselguhr, tricalcium phosphate, powdered cork or adsorbent carbon black; water-soluble polymers, resins, waxes; or solid fertilizers. Such compositions can, if so desired, contain one or more compatible agents such as wetting agents, dispersing agents, emulsifiers or dyes which, when they are solid, can also act as diluents. Where the additives are alkaline and will likely increase the pH of the compositions, e.g., talc, lime, calcium carbonate, and marble, the amount by which they are added should not cause the pH to exceed the claimed ranges or additional acid should be added to maintain the desired pH. Preferably, such materials should be avoided altogether.
The fillers can also be liquids, for example: water, alcohols, in particular butanol or glycol, as well as ethers or esters thereof, in particular methyl glycol acetate; ketones, in particular acetone, cyclohexanone, methyl ethyl ketone, methyl isobutyl ketone or isophorone; petroleum fractions such as paraffinic or aromatic hydrocarbons, in particular xylenes or alkylnaphthalenes; mineral or plant oils; aliphatic chlorohydrocarbons, in particular trichloroethane or methylene chloride; aromatic chlorohydrocarbons, in particular chlorobenzenes; water-soluble or highly polar solvents such as dimethylformamide, dimethyl sulphoxide, N,N-dimethylacetamide or N-methylpyrrolidone; N-octylpyrrolidone, liquefied gases; or the like, whether they are taken separately or as a mixture.
As mentioned above, depending upon the end-use application, the inventive bioactive agrichemical compositions or formulations will contain one or more additional surfactants (additional to the surfactant(s) that are optionally part of the bioactive acid solution or bioactive acid composition) as emulsifiers, dispersing agents, wetting agents and the like. These additional surfactants may be cationic, anionic, nonionic or amphoteric surfactants or mixtures of these surfactants. Among those surfactants which are used, for example, are polyacrylic acid salts, lignosulphonic acid salts, phenolsulphonic or naphthalenesulphonic acid salts, polycondensates of ethylene oxide with fatty alcohols or fatty acids or fatty esters or fatty amines, substituted phenols (in particular alkylphenols or arylphenols), ester-salts of sulphosuccinic acid, taurine derivatives (in particular alkyl taurates), phosphoric esters of alcohols or of polycondensates of ethylene oxide with phenols, fatty acid esters with polyols, or sulphate, sulphonate or phosphate functional derivatives of the foregoing compounds as well as those surfactants described above relative to the synergistic surfactant for the bioactive composition. Here, however, the surfactants are generally present at much higher concentrations versus that needed to show synergy with respect to the acid/metal combination. The presence of at least one additional surfactant is generally essential when the active materials and/or the inert filler are insoluble or only sparingly soluble in water and when the filler for the said composition to be applied is water. For foliar applications, the choice of surfactants is oftentimes paramount for obtaining good bioavailability of the active material(s); thus, a combination of a surfactant of hydrophilic nature (HLB>10) and a surfactant of lipophilic nature (HLB<5) will preferably be used.
In agricultural applications as well as in applications where it is desired to affix the bioactive composition to a surface, the compositions will typically have a binder, rain fastener, or other adhesive type components. Suitable binders are well known and include, e.g., water-soluble and water-dispersible film-forming polymers. Suitable polymers have an average molecular weight of at least about 1,000 up to about 100,000; more specifically at least about 5,000, up to about 100,000. The aqueous compositions generally contain from about 0.5% to about 10%, preferable from about 1.0% to about 5%, by weight of the composition of the binder, film-forming polymer and the like. Suitable film-forming polymers include, but are not limited to a) alkyleneoxide random and block copolymers such as ethylene oxide-propylene oxide block copolymers (EO/PO block copolymers) including both EO-PO-EO and PO-EO-PO block copolymers; ethylene oxide-butylene oxide random and block copolymers, C2-C6 alkyl adducts of ethylene oxide-propylene oxide random and block copolymers, C2-C6 alkyl adducts of ethylene oxide-butylene oxide random and block copolymers; b) polyoxyethylene-polyoxypropylene monoalkylethers such as methyl ether, ethyl ether, propyl ether, butyl ether or mixtures thereof; c) vinylacetate/-vinylpyrrolidone copolymers, d) alkylated vinylpyrrolidone copolymers, e) polyvinylpyrrolidone, and f) polyalkyleneglycol including the polypropylene glycols and polyethylene glycols. Specific examples of suitable polymers include Pluronic P103 (BASF) (EO-PO-EO block copolymer), Pluronic P65 (BASF) (EO-PO-EO block copolymer), Pluronic P108 (BASF) (EO-PO-EO block copolymer), Vinamul 18160 (National Starch) (polyvinylacetate), Agrimer 30 (ISP) (polyvinylpyrrolidone), Agrimer VA7w (ISP) (vinyl acetate/vinylpyrrolidone copolymer), Agrimer AL 10 (ISP) (alkylated vinylpyrrolidone copolymer), PEG 400 (Uniqema) (polyethylene glycol), Pluronic R 25R2 (BASF) (PO-EO-PO block copolymer), Pluronic R 31R1 (BASF) (PO-EO-PO block copolymer) and Witconol NS 500LQ (Witco) (butanol PO-EO copolymer).
Additional adhesive and adhesive type materials that may be used include carboxymethylcellulose, or natural or synthetic polymers in the form of powders, granules or matrices, such as gum arabic, latex, polyvinylpyrrolidone, polyvinyl alcohol or polyvinyl acetate, natural phospholipids, such as cephalins or lecithins, or synthetic phospholipids can be used in the formulations.
It may also be desirable to thicken the bioactive compositions and formulations, especially where there is concern that the composition will quickly run off or run down the substrate to which it is applied. Suitable thickeners include water-soluble polymers which exhibit pseudoplastic and/or thixotropic properties in an aqueous medium such as gum arabic, gum karaya, gum tragacanth, guar gum, locust bean gum, xanthan gum, carrageenan, alginate salt, casein, dextran, pectin, agar, 2-hydroxyethyl starch, 2-aminoethyl starch, 2-hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose salt, cellulose sulfate salt, polyacrylamide, alkali metal salts of the maleic anhydride copolymers, alkali metal salts of poly(meth)acrylate, and the like. As suitable thickeners, including thixotropes, there may also be mentioned attapulgite-type clay, silica, fumed silica, carrageenan, croscarmellose sodium, furcelleran, glycerol, hydroxypropyl methylcellulose, polystyrene, vinylpyrrolidone/styrene block copolymer, hydroxypropyl cellulose, hydroxypropyl guar gum, and sodium carboxymethylcellulose. Xanthan gum is preferred.
In the case of bioactive agrichemical compositions that are or may be subject to freezing during storage or use, especially aqueous and aqueous-based concentrates and solutions, it is desirable to add antifreeze additives. Specific examples of suitable antifreezes include ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,4-pentanediol, 3-methyl-1,5-pentanediol, 2,3-dimethyl-2,3-butanediol, trimethylol propane, mannitol, sorbitol, glycerol, pentaerythritol, 1,4-cyclohexanedimethanol, xylenol, bisphenols such as bisphenol A or the like. In addition, ether alcohols such as diethylene glycol, triethylene glycol, tetraethylene glycol, polyoxyethylene or polyoxypropylene glycols of molecular weight up to about 4000, diethylene glycol monomethylether, diethylene glycol monoethylether, triethylene glycol monomethylether, butoxyethanol, butylene glycol monobutylether, dipentaerythritol, tripentaerythritol, tetrapentaerythritol, diglycerol, triglycerol, tetraglycerol, pentaglycerol, hexaglycerol, heptaglycerol, octaglycerol and the like. As a particular subset of suitable antifreeze materials there can be mentioned ethylene glycol, propylene glycol and glycerin.
It is possible to use dyes such as inorganic pigments, such as, for example: iron oxides, titanium oxides, Prussian blue; organic dyestuffs, such as those of the alizarin, azo or metal phthalocyanin type; or of trace elements such as iron, manganese, boron, copper, cobalt, molybdenum or zinc salts. The use of such dyes enables one to determine which areas and substrates, including plants, have been treated with the bioactive composition. Such marking is especially important for a variety of applications and reasons. For example, the use of dyes in seed treatments will enable a quick visual determination of which seeds have and have not been treated. Similarly, in disinfectant applications, for example in a biotechnology laboratories, microbiology laboratories, food and/or pharmaceutical manufacturing and processing facilities and the like, the use of they dye, will allow those performing the cleaning operation to ensure that all surfaces are treated. Here, for example, the material could be applied and allowed to sit for a brief period before being wiped to leave the cleaned surface. Further, in aerial, drop or broadcast application, it enables the pilot or driver of the dispensing vehicle see what areas have already been treated.
Although not all additives and adjuvants have been described above, those skilled in the art, particularly in the art pertinent to the specific end-use application anticipated, will certainly appreciate what other ingredients, additives and the like would or should be used for their application. The amount by which each additive is to be incorporated into the compositions will, once again, depend upon the end-use application and the method of application and environment into which it is to be employed. Generally, though, the selection and amount is that which is conventional for such additives in such applications. However, with the selection of any additives, it is important to ensure that they will not interfere with the bioactivity of the compositions of the present invention or that any such interference will be minimized so as to enable one to take the most advantage of the bioactive compositions of the present invention. Those skilled in the art, based upon the teachings set forth herein and in the following examples, will appreciate where attention is due and, in any event, such can be addressed by simple screening applications.
As noted above, it is important to avoid the use of conventional bioactive agrichemical actives as well as any other additives and components, including those of the types mentioned above, that interfere with or adversely affect the bioefficacy of the compositions according to the present invention. Most especially, it is important to avoid the use of those agrichemical actives and other additives or compounds that are known to or will likely irreversibly or strongly sequester, bind, or complex with the antimicrobial metal ions in solution. In following, not intending to be bound by theory, it is believed that retention of the antimicrobial metal ionic charge is important for maintaining bioefficacy. For example, especially with respect to copper ions, it is best to avoid the use of ammonium salts such as ammonium sulphate, ammonium chloride, ammonium citrate, ammonium phosphate. To the extent any such materials are present or to be used, their use or, more accurately, the amount thereof, should be minimized and/or the metal ion concentration increased to offset the loss of free ions in solution compounds.
The compositions of the present invention may be made by any known method for formulating agrichemical compositions, especially antimicrobial and antifungal type compositions. Generally speaking, whether making a concentrate or the application ready bioactive agrichemical compositions of the present invention or whether making a liquid system or a solid system, the bioactive acid solution or, if applicable, the solid bioactive acid composition is prepared before the addition of a conventional bioactive agrichemical active or formulation and/or other conventional agrichemical additives and agents.
The bioactive acid solution may be prepared in a number of conventional ways. For example, each component may be dissolved in the appropriate solvent, most notably water or a water-based solvent, and the solutions combined in the appropriate proportions. To some extent, the sequence of the addition and whether a pre-concentrate of the acid in the solvent is formed depends upon the solubility of the solids themselves. Preferably, the acid is initially dissolved in the appropriate solvent to the desired concentration. Where one is intending to form a concentrate, the amount of acid to be dissolved in the solvent should be such that the acid concentration is at least 40 percent and preferably form 40 to 80 percent. The antimicrobial metal ion source or sources are then dissolved in the concentrated acidic solution. This method may also be used in preparing a non-concentrated bioactive agrichemical composition where the rate at which the antimicrobial metal ion source or sources dissolves is increased with higher acid concentration. For example, as mentioned above, where the metal ion source is an antimicrobial metal ion containing ion-exchange type agent, especially those whose core is a zeolite, the use of concentrated acids has been found to readily dissolve the zeolite. Thereafter, the concentrated solution is merely diluted to the desired concentration after the solids are dissolved.
Where there is difficulty in dissolving the antimicrobial metal source or sources in the concentrated or dilute acid solution, or the rate is undesirably slow, the antimicrobial metal ion source or sources may first be dissolved in water or another suitable aqueous-based solvent and that combined with the formed acid solution. Here, the acid solution is preferably of a higher concentration than intended in the bioactive acid solution so as to account for the dilution upon adding the dissolved antimicrobial metal ion source or sources.
Similarly, whether preparing concentrates or final, end-use formulations, it may be desirable to make individual stock solutions of each of the components of the bioactive acid solution which stock solutions are then combined in the appropriate proportions. Again, the concentration of each stock solution would be tailored to account for the dilution upon their combination. Obviously, for forming concentrates, the stock solutions will typically be of higher concentration than might otherwise be necessary if using the stock solutions for preparing the final, end-use diluted formulations.
In each of the foregoing instances, the solvent/solutions may be heated and are preferably agitated to expedite the dissolving of the solids in the liquid system. Furthermore, while the dissolution of antimicrobial metal ion source or sources is perhaps the simplest and most cost effective method of the preparation of the bioactive acid solutions, these bioactive acid solutions may also be prepared by, e.g., electrolytically generating the metal ion in acid solutions as seen in Arata et. al. (U.S. Pat. No. 6,197,814; US 2003/0198689A1, US 2003/0178374A1; US2005/0245605A1 and US2006/0115440A1, all of which are incorporated herein by reference in their entirety) or by high temperature and pressure as seen in Cummins et. al. (U.S. Pat. No. 7,192,618, incorporated herein be reference).
The surfactants may be added to the bioactive acid solution or the concentrate or may be added concurrent with or subsequent to the combination of the bioactive acid solution with the conventional bioactive agrichemical composition.
When desiring to make a liquid bioactive acid solution concentrate, one may prepare the highly concentrated solution as discussed above or make a somewhat diluted form which is then further concentrated by allowing some of the solvent to evaporate. This is particularly beneficial where the antimicrobial metal ion source or sources and/or the surfactants and/or other constituents are not soluble in and/or or are not sufficiently and/or expeditiously dissolved in the acid solution.
Depending upon the ultimate form of the inventive bioactive agrichemical composition, it may likewise be desirable to prepare a solid bioactive acid composition concentrate. These solid bioactive acid composition concentrates may also be made in a number of ways. For example, the acid, the antimicrobial metal ion source or sources and, if present, the surfactant can be dry blended. Dry blending is still possible even if the surfactant or one of the surfactants is a liquid since the amount employed is so low and will be adsorbed or absorbed by the dry materials. The dry blended materials may be employed as is or are preferably compressed to form granules. Alternatively, the solid bioactive acid composition concentrate can be formed by first preparing the bioactive acid solution concentrate mentioned above, using a volatile solvent, e.g., water or a water-base solvent, and then allowing the solvent to evaporate to leave the solid material. As necessary, the solid material is then crushed or ground to form small particles, powder or granules, of the solid bioactive acid composition.
The solid bioactive acid composition concentrate may be used to form the liquid bioactive acid solution as a concentrate or as its final, end-use diluted form. In the former, the solid concentrate is dissolved in as minimal a volume of an appropriate solvent, notably water or a water based solvent, to form the concentrate. If need be or desired, especially if dissolving is hastened, a larger volume of the solvent may be employed and then partially evaporated to concentrate the materials.
Solid bioactive acid compositions in their final, end-use dilution may be prepared by dry blending the acid, the antimicrobial metal ion source or sources and the surfactant with a solid filler material or the aforementioned solid bioactive acid composition concentrate may be let down or diluted with solid filler materials. Alternatively, and preferably, the solid bioactive acid composition is prepared by treating a filler material with a bioactive acid solution. Here the liquid bioactive acid solution is applied to or combined with the filler material, which is preferably in particle form, and is adsorbed by and/or absorbed by the particles of the filler. For example, a mist of the bioactive acid solution may be sprayed or a steady or intermittent stream of the bioactive acid solution may be poured onto the particles as they are tumbled, stirred, etc. This embodiment has the added advantage that the amount or concentration of liquid acid solution applied to the adsorbent or absorbent carrier or conventional bioactive agrichemical active for formulated active can be higher than would be applied in the liquid diluted state so as to allow for longer term bioefficacy. In essence, the treated carrier or conventional active serves as a reservoir of the bioactive components of the liquid acid solution.
Given the high transportation costs and the ease of dilution, it is most preferable and cost effective to prepare concentrates, especially liquid concentrates, of the inventive bioactive agrichemical compositions which concentrates are then diluted or let down at the time of application. These liquid concentrates are then diluted or let down with an appropriate solvent, especially water or a water based solvent, to the desired concentration for application.
The bioactive agrichemical compositions of the present invention have a myriad of agricultural and horticultural applications including as fungicides, bactericides, and/or plant, stramenophile and fungi-like protisticides and may be applied to seeds, soils, plants, trees, and the like. These compositions show particular promise as fungicides, bactericides, and plant, stramenophile and fungi-like protisticides due to their unique and surprising strong bioefficacy at extremely low levels of antimicrobial metals. For example, the use of these materials allows for effective rates of applications wherein, for example, the amount of copper to be applied is on the order of grams per acre, not kilograms as is necessary with conventional copper and copper based fungicides.
Besides the marked bioefficacy at such low levels of antimicrobial metal ion, another attribute of the inventive bioactive agrichemical compositions is that they have no or little phytotoxicity. This is especially important since a bioeffective material that severely damages or kills the plant concurrent with killing the target organism is of little use unless one is not concerned with losing a crop and is most interested in controlling the target organism before it gets out of control. Another important and beneficial factor associated with the inventive compositions is the fact that they do not and are not likely to induce or be associated with any resistance in the target organisms. This contrasts sharply with the use of organic bioactive agrichemicals, especially fungicides and antibiotics, for which studies and actual commercial practice has shown a marked and growing tendency of resistance among the targeted organisms, even within a few years or less of their first use. The development of such bioactive agrichemical resistant microorganisms, while bothersome at the present time, could lead to catastrophic results if unchecked.
Typically, the rate of application of the inventive bioactive agrichemical compositions of the present invention is such that the total amount of antimicrobial metal ions (as metal) originated from the dissolved antimicrobial metal ion source or sources applied per acre will be about 200 grams or less, preferably 100 grams or less, more preferably 50 grams or less, most preferably 20 grams or less. Of course the specific application rate and, thus, the total amount applied per acre, will vary from target organism to target organism, from one form to another and from one application method to another. Indeed, suitable rates may be such that the total metal ion (as metal) may be on the order of 5 grams per acre, even on the order of fractions of a gram per acre, perhaps as low as 0.5 grams per acre or even 0.05 grams per acre. While higher loadings, higher than 200 grams per acre, may provide even greater or faster bioefficacy, the trade-off of increased environmental, health and safety concerns does not generally warrant or is not typically justified by the increased, oftentimes nominal increase, in bioefficacy.
The bioactive agrichemical compositions of the present invention may be applied to any number of agricultural, including horticultural, crops including ornamental plants, shrubs and trees; flowering plants; fruiting trees, vegetable crops; feed crops; ornamental grasses and turf; etc. Exemplary crops that are of particular concern due to their significant economic and food source impact include soy beans, tomatoes, potatoes, apples, peanuts, grapes, almonds, sugarbeets and citrus. Diseases and microorganisms to be targeted by the bioactive agrichemical compositions of the present invention include, but are not limited to citrus canker; soybean rust; leaf, stem and stripe rusts; leaf blights; early blights; late blights; fire blight; leaf spots; powdery mildew; bacterial canker; early rot; Alternaria leaf blight; Alternaria leaf spot; Fabrea leaf spot; bacterial wilt; Pierce's disease; Karnal bunt; citrus greening; potato wart; Agrobacterium tumefaciens; clavibacter michiganensis; Pseudomonas syrinhea; fusarium; phytophthora infestans; Alternaria solani; Erwinia amylovora; Botrytis cinerea; Xanthomonas vesicatoria; and the like. A more comprehensive listing of specific pathogens and the crops they attack are set forth in Tate—US 2005/0079227A1, the contents of which are hereby incorporated herein by reference.
These compositions may be applied in any conventional manner, spraying, dusting, spreading, etc., as also noted above. Typically any given formulation will be applied in the manner consistent for the targeted crop and microorganism. Furthermore, it is also contemplated that any conventional bioactive agent or other agrichemical additive, if any, to be used may be applied individually, concurrently or sequentially (essentially as a two-part system), within a few hours of each other, preferably within an hour or two of each other, particularly where there is concern that the conventional bioactive agent or other agrichemical additive may interfere with the performance of the bioactive acid solution or composition of the present invention, e.g., adversely sequester or bind the antimicrobial metal ions. Typically though, especially for convenience and cost savings, the inventive agrichemical compositions of the present invention will be applied as a single composition.
Unlike disinfectants where bioefficacy is measured in terms of log kill, particularly within specified time period, the bioefficacy of the bioactive agrichemical compositions of the present invention is more so represented or evidenced by an increase in yields or reduction in loss of the crop. Even a 10% improvement in yield can have a significant economic impact. In essence, even a seemingly minor reduction in the target organism or a modest inhibition in the growth or proliferation of the target organism can manifest an acceptable bioefficacy. Furthermore, the duration of this effect need not be long-lived, for example, efficacy over a couple of days or so may be sufficient. Generally, and preferably, it is desirable to see a significant reduction, 25% or more, preferably 50% or more, in the growth or proliferation of the target organism over two or more, preferably four or more days. More preferably, it is desirable to see a 85% or more, most preferably a 95% or more, reduction in the growth or proliferation of the target organism over two or more, most preferably four or more days. Again, though, from a commercial perspective, the desired outcome is an increase, at least a 10% increase, preferably at least a 30% increase, most preferably a 65% increase, in yield as compared to the untreated crop.
The following examples are presented as demonstrating the bioefficacy of the bioactive agrichemical compositions according to the present invention as well as the unexpected synergy resulting from the use of the bioactive acid solutions or bioactive acid compositions in combination with conventional bioactive agrichemical actives and formulated actives—the latter being the subject matter of the aforementioned International and United States co-pending and co-filed patent applications. These examples are merely illustrative of the invention and are not to be deemed limiting thereof. Those skilled in the art will recognize many variations that are within the spirit of the invention and scope of the claims.
Saccharomycetes Cerevisiae Studies
A series of experiments (Examples 1-269 below) were conducted to evaluate the performance of the individual components of the claimed bioactive compositions as well as various combinations thereof, including, the claimed compositions themselves, in suppressing the growth of Saccharomycetes Cerevisiae (Fleishmann's Bakers yeast). Saccharomycetes Cerevisiae was selected as a test organism as it is generally accepted in the industry as an indicator or surrogate organism for a wide variety of fungi. In each of these experiments, the same general procedure was followed unless otherwise indicated.
Experimental Detail: A growth medium was prepared by adding 10 grams of nutrient medium (Difco Sabouraud dextrose broth from BD of Franklin Lakes, N.J., USA) to 300 ml of distilled water. Fleishmann's Bakers yeast was then added to the growth medium while mixing using a magnetic stirrer until a uniform dispersion was obtained having an initial turbidity of between about 50 and 100 NTU as measured using a HF Instruments DRT 100B Turbidity Meter. Once the appropriate dispersion was obtained, 20 ml aliquots were then dispensed, with continued mixing, into 40 ml borosilicate glass vials with Teflon lined caps (VWR International Cat. No. 15900-004). The system/component to be evaluated was then added to the vial and intimately shaken to ensure a good, substantially homogeneous mixture. The turbidity of each mixture was then determined and the vial transferred to an incubator at 30° C. Each vial was periodically removed from the incubator and the mixture in the vials assessed for turbidity: the specific timing for such evaluation was as set forth in the discussion of the experiments and the accompanying tables.
In each experiment, unless otherwise specified, a 2 ml aqueous solution containing the specified bioactive system or component thereof was added to the 20 ml yeast suspension and mixed thoroughly. Typically the surfactants were added separately in a concentrated solution in water; however, the volume added was negligible: a fraction of an ml. For convenience in understanding efficacy levels, the amounts or concentrations of the various components presented in each of the following tables and experiments are of the diluted material in the test vial: not of the concentrate added to the test vial. Furthermore, the concentrations presented are on the basis of a 20 ml total volume, not the actual 22+ ml volume. Multiplying each of the listed concentrations by 0.9 (or 0.95 with those compositions using 1 ml aqueous solutions) will provide a more accurate assessment of the concentrations of the various components evaluated, i.e., a 5 ppm silver concentration is actually closer to 4.5 ppm. Finally, for those vials to which no bioactive system or component thereof was added (the controls) or which only contained the surfactants, 2 ml of additional growth medium was added to ensure relative equivalent dilutions of the yeast.
In the tables below, the results are presented as the actual turbidity readings (NTU) with a sub-table presenting the change or delta in NTU values. Given the nature of the system, changes in turbidity are reflective of the relative performance/bioefficacy of the bioactive systems and their components. In certain instances, a high level of bioactive material, especially the metal component, caused an immediate and relatively sharp increase in optical density or turbidity. This was believed to have been a result of lysing of at least a portion on the yeast cells themselves. Consequently, especially in those examples having a high level of bioactive, it is equally, if not more, important to look at the change in turbidity from either the half hour or one hour turbidity results, if presented, forward, not from time zero.
EXAMPLES 1-21
Acid Concentration
A first series of experiments was conducted for evaluating the performance of various antimicrobial metals and combinations of such metals, with and without citric acid and with and without sodium lauroyl sarcosinate anionic surfactant. Each of the metals was added in the form of an aqueous solution of their citrate salts, namely, silver citrate, copper citrate and zinc citrate, or, in the case of Examples 16-19, as a mixture of all three citrate salts (MI1). The specific formulations evaluated and the resultant yeast growth study results are shown in Tables 1 and 1A.











TABLE 1










Na




Metal Ion
Citric
Lauroyl



Ex-
and
Acid
Sarcos-
Turbidity (NTU)















am-
Amount
(wt
inate
Time
Time
T 18
T 24
T 96


ple
(ppm)
%)
(wt %)
zero
1 Hr
hours
hours
Hours


















1
Ag 5 ppm
0

44.5
59.6
890
932
995


2
Ag 5 ppm
1

47.5
64
882
902
1044


3
Ag 5 ppm
2

50.9
68.4
881
950
1025


4
Ag 5 ppm
0
0.005
46.8
51.5
596
677
673


5
Ag 5 ppm
1
0.005
59.4
68.4
85
130
854


6
Ag 5 ppm
2
0.005
70.9
75
85
120
880


7
Zn 5 ppm
0

43.8
64.5
992
993
1051


8
Zn 5 ppm
1

46.6
66.5
934
962
1027


9
Zn 5 ppm
2

49.5
71
936
1038
1063


10
Zn 5 ppm
0
0.005
45.9
63
656
747
712


11
Zn 5 ppm
1
0.005
57
71
160
223
744


12
Zn 5 ppm
2
0.005
73
76.5
105
119
466


13
Cu 5 ppm
0

45.6
68
940
1021
1100


14
Cu 5 ppm
1

49
72
940
1018
1102


15
Cu 5 ppm
2

49
74
900
973
1100


16
MI1
0
0.005
39
44.5
449
575
658


17
MI1
1
0.005
73.9
87
100
105
732


18
MI1
2
0.005
132
137
137
137
690


19
MI1
1
0.01
74.5
74.8
87
89
116


20
Control


53.2
69.4
1031
1085
1122



(No










Biocide)









22
Control


53.2
78
1101
1093
1128



(No










Biocide)





* MI1 a 4% citric acid solution containing of 50 ppm each of Ag, Cu and Zn per ml giving ~5 ppm of each in the test vial

















TABLE 1A










Na




Metal Ion
Citric
Lauroyl
Change in Turbidity from T0 (delta



and
Acid
Sarcos-
NTU)














Ex-
Amount
(wt
inate
Time
T 18
T 24
T 96


ample
(ppm)
%)
(wt %)
1 Hr
hours
hours
Hours

















1
Ag 5 ppm
0

15.1
845.5
887.5
950.5


2
Ag 5 ppm
1

16.5
834.5
854.5
996.5


3
Ag 5 ppm
2

17.5
830.1
899.1
974.1


4
Ag 5 ppm
0
0.005
4.7
549.2
630.2
626.2


5
Ag 5 ppm
1
0.005
9
25.6
70.6
794.6


6
Ag 5 ppm
2
0.005
4.1
14.1
49.1
809.1


7
Zn 5 ppm
0

20.7
948.2
949.2
1007.2


8
Zn 5 ppm
1

19.9
887.4
915.4
980.4


9
Zn 5 ppm
2

21.5
886.5
988.5
1013.5


10
Zn 5 ppm
0
0.005
17.1
610.1
701.1
666.1


11
Zn 5 ppm
1
0.005
14
103
166
687


12
Zn 5 ppm
2
0.005
3.5
32
46
393


13
Cu 5 ppm
0

22.4
894.4
975.4
1054.4


14
Cu 5 ppm
1

23
891
969
1053


15
Cu 5 ppm
2

25
851
924
1051


16
MI1
0
0.005
5.5
410
536
619


17
MI1
1
0.005
13.1
26.1
31.1
658.1


18
MI1
2
0.005
5
5
5
558


19
MI1
1
0.01
0.3
12.5
14.5
41.5


20
Control


16.2
977.8
1031.8
1068.8



(No









Biocide)








21
Control


24.8
1047.8
1039.8
1074.8



(No









Biocide)





* MI1 a 4% citric acid solution containing of 50 ppm each of Ag, Cu and Zn per ml giving ~5 ppm of each in the test vial






As seen in Tables 1 and 1A, those formulations having both the acid and the anionic surfactant provided marked yeast growth inhibition through at least the first 24 hour period, even with the low lever of anionic surfactant. Those samples with just the metal ion or the metal ion in combination with the acid had no appreciable effect on yeast growth. Although some inhibition was also noted in those samples wherein only the metal(s) and surfactant were present, the inhibition was not appreciable. Rather, as noted, the further presence of excess acid gave a marked and unexpected level of improvement. Finally, that formulation having all three antimicrobial metal ions, plus the acid and surfactant provided continued to show excellent yeast growth inhibition even at the 96 hour test limit.
EXAMPLES 22-42
Surfactant Evaluation
A similar series of experiments was conducted again to evaluate the performance of various combinations of the components of the bioactive compositions of the present invention as well as to demonstrate other anionic surfactants and combinations of surfactants. The specific formulations evaluated and the yeast growth results are presented in Tables 2 and 2A.
Once again, the importance of all three constituents was evident from the results shown in Tables 2 and 2A. These results further confirm that even a low excess acid content, here 0.4%, provides excellent inhibition in yeast growth through 96 hours. The somewhat less than ideal results shown in Examples 26 and 29 suggest some variation amongst anionic surfactants, at least with sodium lauryl sulfate (SLS), with zinc and copper ions. However, the results are still significantly better than without a surfactant at all and suggest a possible synergy with two. Furthermore, because of the easier solubility of the SLS, as compared to the sodium lauroyl sarcosinate (NaLS), the presence of the SLS helps improve and/or enhance the solubility of the NaLS under acid conditions.
EXAMPLES 43-57
Low Concentration Evaluation
A series of experiments were conducted again to evaluate the performance of various combinations of the components of the bioactive compositions of the present invention, this time focusing on the impact of the low concentrations of the components and their combinations. In this set of experiments, 1 ml aqueous solutions of the bioactive/citric acid components were added to the 20 ml vials. The specific formulations evaluated and the yeast growth results are presented in Tables 3 and 3A.
As seen in Tables 3 and 3A, once again the combination of bioactive metal ions, citric acid and anionic surfactant demonstrated a marked inhibition in yeast growth as compared to the individual components, even at the low concentrations of excess acid and surfactant. Though, once again, the surfactants appeared to have a marginal inhibitory effect, as compared to the controls, on their own, the inhibition was negligible as compared to that of the systems according to the present invention.










TABLE 2








Metal citrates (ppm) in

Turbidity (NTU)














Example
.4% citric acid
Surfactant* (wt %)
Time zero
T1 hour
T18 hours
T24 hours
T96 Hrs

















22
Copper 5 ppm

103
114
410
463
588


23
Zinc 5 ppm

103
118
475
488
589


24
Silver 5 ppm

155
168
181
190
670


25
Copper 5 ppm
.005 NaLS
145
146
157
160
149


26
Copper 5 ppm
.005 SLS
119
128
252
326
502


27
Copper 5 ppm
.005 NaLS:.005 SLS
145
144
156
154
157


28
Zinc 5 ppm
.005 NaLS
148
156
157
157
157


29
Zinc 5 ppm
.005 SLS
126
134
217
234
539


30
Zinc 5 ppm
.005 NaLS:.005 SLS
155
155
157
157
158


31
Silver 5 ppm
.005 NaLS
170
170
184
184
180


32
Silver 5 ppm
.005 SLS
177
177
193
196
196


33
Silver 5 ppm
.005 NaLS:.005 SLS
193
190
198
199
199


34
Copper 2.5 ppm:Zinc 2.5 ppm

99
109
498
510
614


35
Copper 2.5 ppm:Silver 2.5 ppm

128
152
424
530
727


36
Zinc 2.5 ppm:Silver 2.5 ppm

128
151
541
621
720


37
Control 1 (no biocide)

91
114
560
580
754


38
Control 2 (no biocide)

91
114
563
584
726


39
Copper 2.5 ppm:Zinc 2.5 ppm
.005 NaLS:.005 SLS
192
180
193
193
193


40
Copper 2.5 ppm:Silver 2.5 ppm
.005 NaLS:.005 SLS
181
204
205
206
206


41
Zinc 2.5 ppm:Silver 2.5 ppm
.005 NaLS:.005 SLS
194
193
212
212
212


42
Copper 2.5 ppm:Silver 2.5 ppm:Zinc
.005 NaLS:.005 SLS
193
193
199
200
205



2.5 ppm





*NaLS—sodium lauroyl sarcosinate, SLS—sodium lauryl sulfate
















TABLE 2A








Metal citrates (ppm) in

Change in Turbidity from T0 (delta NTU)













Example
.4% citric acid
Surfactant* (wt %)
T1 hour
T18 hours
T24 hours
T96 Hrs
















22
Copper 5 ppm

11
307
360
485


23
Zinc 5 ppm

15
372
385
486


24
Silver 5 ppm

13
26
35
515


25
Copper 5 ppm
.005 NaLS
1
12
15
4


26
Copper 5 ppm
.005 SLS
9
133
207
383


27
Copper 5 ppm
.005 NaLS:.005 SLS
−1
11
9
12


28
Zinc 5 ppm
.005 NaLS
8
9
9
9


29
Zinc 5 ppm
.005 SLS
8
91
108
413


30
Zinc 5 ppm
.005 NaLS:.005 SLS
0
2
2
3


31
Silver 5 ppm
.005 NaLS
0
14
14
10


32
Silver 5 ppm
.005 SLS
0
16
19
19


33
Silver 5 ppm
.005 NaLS:.005 SLS
−3
5
6
6


34
Copper 2.5 ppm:Zinc 2.5 ppm

10
399
411
515


35
Copper 2.5 ppm:Silver 2.5 ppm

24
296
402
599


36
Zinc 2.5 ppm:Silver 2.5 ppm

23
413
493
592


37
Control 1 (no biocide)

23
469
489
663


38
Control 2 (no biocide)

23
472
493
635


39
Copper 2.5 ppm:Zinc 2.5 ppm
.005 NaLS:.005 SLS
−12
1
1
1


40
Copper 2.5 ppm:Silver 2.5 ppm
.005 NaLS:.005 SLS
23
24
25
25


41
Zinc 2.5 ppm:Silver 2.5 ppm
.005 NaLS:.005 SLS
−1
18
18
18


42
Copper 2.5 ppm:Silver 2.5 ppm:Zinc
.005 NaLS:.005 SLS
0
6
7
12



2.5 ppm



















TABLE 3








Bioactive

Turbidity(NTU)















Example
Metal*
Citric Acid (wt %)
Surfactant** (wt %)
OD (To)
OD (T1 hr)
OD (T18)
OD (T24)
OD (T48)


















43


0.01 NaLS
43
45
550
613
521


44


0.02 NaLS
43
40
460
524
624


45


0.01 SLS
43
47
675
728
758


46


0.02 SLS
37
42
495
610
605


47


0.01 NaLS/0.01 SLS
40
41
370
466
580


48


0.005 NaLS/0.005 SLS
43
47
630
696
726


49

0.05

42
46
835
920
878


50

0.1

38
44
780
864
852


51
MI1
0.2

50
62
809
891
915


52
MI1
0.2
0.01 NaLS
64
63
67
68
69


53
MI1
0.2
0.01 SLS
61
65
300
569
1039


54
MI1
0.2
0.005 NaLS/0.005 SLS
60
63
62
63
73


55
MI1
0.2
0.01 NaLS/0.01 SLS
85
76
76
79
79


56
Control 1


43
51
960
997
939


57
Control 2


43
51
890
986
887





*MI1 a 4% citric acid solution containing of 50 ppm each of Ag, Cu and Zn per ml giving (@ 1 ml) ~5 ppm of each in the test vial


**NaLS—sodium lauroyl sarcosinate, SLS—sodium lauryl sulfate














TABLE 3A








Change in Turbidity from T0 (delta NTU)














Example
Bioactive Metal*
Citric Acid (wt %)
Surfactant** (wt %)
OD (T1 hr)
OD (T18)
OD (T24)
OD (T48)

















43


0.01 NaLS
2
507
570
478


44


0.02 NaLS
−3
417
481
581


45


0.01 SLS
4
632
685
715


46


0.02 SLS
5
458
573
568


47


0.01 NaLS/0.01 SLS
1
330
426
540


48


0.005 NaLS/0.005 SLS
4
587
653
683


49

0.05

4
793
878
836


50

0.1

6
742
826
814


51
MI1
0.2

12
759
841
865


52
MI1
0.2
0.01 NaLS
−1
3
4
5


53
MI1
0.2
0.01 SLS
4
239
508
978


54
MI1
0.2
0.005 NaLS/0.005 SLS
3
2
3
13


55
MI1
0.2
0.01 NaLS/0.01 SLS
−9
−9
−6
−6


56
Control 1


8
917
954
896


57
Control 2


8
847
943
844





*MI1 a 4% citric acid solution containing of 50 ppm each of Ag, Cu and Zn per ml giving (@ 1 ml) ~5 ppm of each in the test vial


**NaLS—sodium lauroyl sarcosinate, SLS—sodium lauryl sulfate






EXAMPLES 58-71
Ion-Exchange Metal Ion Source
A metal citrate solution was prepared by adding approximately 4 grams of citric acid to about 8 grams of water and mixed until fully dissolved. Thereafter, 0.1 grams each of two ion-exchange type antimicrobial agents, AgION AC10D and AgION AK10D antimicrobial agents from AgION Technologies of Wakefield, Mass., USA, were added to the concentrated citric acid solution with agitation until the antimicrobial agents fully dissolved. Approximately 92 grams of water was then added to provide a 4% citric acid solution having dissolved therein 0.1 wt % AC10D and 0.1 wt % AK10D. AgION AK10D contains about 5.0% by weight silver and about 13% by weight zinc and AgION AC10D contains about 6.0% by weight copper and about 3.5% by weight silver. Various quantities of the so formed citric acid solution were then added to test vials so as to provide a silver content in the test vials of approximately 1.25 ppm, 2.5 ppm, 5.0 ppm and 10 ppm. Additionally, different surfactant and surfactant combinations were added to certain vials to demonstrate the effect of different metal and acid contents on bioefficacy with and without surfactants. The specific formulations evaluated and the yeast growth results are presented in Tables 4 and 4A.
As seen in Tables 4 and 4A, the compositions according to the present invention provided marked inhibition in yeast growth. Although Example 61 containing the higher concentration of metal ions (10 ppm silver, 7 ppm copper and 15.3 ppm zinc), showed good yeast growth inhibition, the higher degree of efficacy comes with the concomitant increase in the release of these metals into the environment. This becomes especially important where the bioactive materials are to be used in or near marine and/or agricultural applications. Thus, while high metal concentrations, especially of silver, will provide better bioefficacy, they also hasten the impact on aquatic environments. On the other hand, as shown in those examples employing the antimicrobial metal containing acid solutions with the anionic surfactant, especially sodium lauroyl sarcosinate, alone or in combination with sodium lauryl sulfate, the same and even better yeast inhibition is realized with less than half, even less than one-quarter, the metal ion concentrations. Furthermore, these results show that by adjusting the level of surfactant, one may reduce the level of metal ion even more while still providing marked inhibition of the fungi.








TABLE 4








Turbidity (NTU)















Example
Ag Concentration ppm
Surfactant* (wt %)
OD (T zero)
OD (T1 hr)
OD (T18 hr)
OD (T24 hr)
OD (T44 hr)
OD (T120 hr)


















58
1.25

108
128
913
880
954
1136


59
2.5

127
157
865
890
941
1024


60
5

176
199
229
227
234
721


61
10

168
173
191
191
190
180


62
1.25
0.005 NaLS
143
158
240
560
843
708


63
2.5
0.005 NaLS
180
179
204
210
729
843


64
5
0.005 NaLS
194
201
222
221
227
227


65
1.25
0.005 SLS
136
167
953
930
973
1132


66
2.5
0.005 SLS
201
212
880
880
967
1145


67
5
0.005 SLS
248
247
272
272
296
297


68
1.25
.0025 NaLS/.0025 SLS
166
180
343
730
957
986


69
2.5
.0025 NaLS/.0025 SLS
215
217
235
239
759
940


70
5
.0025 NaLS/.0025 SLS
235
235
257
255
259
268


71
Control

101
125
1050
1050
1040
1183





*NaLS—sodium lauroyl sarcosinate, SLS—sodium lauryl sulfate














TABLE 4A








Change in Turbidity (delta NTU)














Example
Ag Concentration ppm
Surfactant* (wt %)
OD (T1 hr)
OD (T18 hr)
OD (T24 hr)
OD (T44 hr)
OD (T120 hr)

















58
1.25

20
805
772
846
1028


59
2.5

30
738
763
814
897


60
5

23
53
51
58
545


61
10

5
23
23
22
12


62
1.25
0.005 NaLS
15
97
417
700
565


63
2.5
0.005 NaLS
−1
24
30
549
663


64
5
0.005 NaLS
7
28
27
33
33


65
1.25
0.005 SLS
31
817
794
837
996


66
2.5
0.005 SLS
11
679
679
766
944


67
5
0.005 SLS
−1
24
24
48
49


68
1.25
.0025 NaLS/.0025 SLS
14
177
564
791
820


69
2.5
.0025 NaLS/.0025 SLS
2
20
24
544
725


70
5
.0025 NaLS/.0025 SLS
0
22
20
24
33


71
Control

24
949
949
939
1082





*NaLS—sodium lauroyl sarcosinate, SLS—sodium lauryl sulfate






Also surprising about this example is the finding that citric acid could dissolve the antimicrobial zeolite particles. This finding presents another means by which the inventive compositions may be made as well as a number of alternative applications for such materials not otherwise possible with the zeolites in their solid form.
EXAMPLES 72-79
Metal Concentration
For this study a concentrated bioactive system (MI2) was prepared comprising a 16% aqueous citric acid solution having dissolved therein silver citrate, copper citrate and zinc citrate, each added in an amount to provide 200 ppm of each metal, together with 0.25% sodium Lauroyl sarcosinate and 0.32% sodium lauryl sulfate. Various amounts of this system were added to the test vials to further assess the impact of metal concentration yeast inhibition. A further example was prepared further including a non-ionic surfactant, Tween 20 (polyoxyethylene (20) sorbitan monolaurate), an emulsifier to assess its impact on performance. The specific formulations evaluated and the results are presented in Tables 5 and 5A.
As seen in Tables 5 and 5A, the high concentrations of metals dramatically inhibited, if not stopped altogether, yeast growth. The solutions of Examples 76, 77 and 78 containing ultra-high metal content appeared to destroy the yeast cells, showing what appeared to be a rapid denaturation of the yeast on addition of the bioactive material to the text vials. It is likely that the initial high turbidity reflected both that arising from the addition of the bioactive materials themselves as well as the destruction of the yeast cells.
Regardless, the results show that marked inhibition is also attained at much lower concentrations of the metal in the presence of the excess acid and surfactant. Indeed, just 15 ppm metals (5 ppm of each) provide excellent inhibition through 82 hours and beyond.










TABLE 5









Concentration



Ex-
MI2*
of



am-
added
each metal
Turbidity (NTU)















ple
(ml)
(ppm)
T0
T18
T22
T24
T64
T82


















72
0
0
63
920
980
964
1020
1050


73
0.1
1
81
608
722
820
1077
1062


74
0.25
2.5
111
126
142
160
752
810


75
0.5
5
145
198
208
208
205
203


76
1.0
10
483
410
395
369
320
300


77
2.0
20
1295
820
714
660
399
264


78
3.0
30
1435
766
620
555
340
340


79
0.5+
5
141
249
405
600
1116
1129





*MI2 a 16% citric acid solution containing of 200 ppm each of Ag, Cu and Zn per ml


+this formulation also contained 0.1 wt % Tween 20 a non-ionic surfactant
















TABLE 5A









Concentration




MI2*
of




added
each metal
Change in Turbidity (delta NTU)














Example
(ml)
(ppm)
T18 − T0
T22 − T0
T24 − T0
T64 − T0
T82 − T0

















72
0
0
857
917
901
957
987


73
0.1
1
527
641
739
996
981


74
0.25
2.5
15
31
49
641
699


75
0.5
5
53
63
63
60
58


76
1.0
10
−73
−88
−114
−163
−183


77
2.0
20
−475
−581
−635
−896
−1031


78
3.0
30
−669.
−815
−880
−1095.
−1095


79
0.5+
5
108
264
459
975
988





*MI2 a 16% citric acid solution containing of 200 ppm each of Ag, Cu and Zn per ml


+this formulation also contained 0.1 wt % Tween 20 a non-ionic surfactant






Finally, the addition of Tween 20 surfactant appeared to be antagonistic to the action of the bioactive systems of the present invention resulting in a reduction in the level of yeast inhibition. Still, this composition (Example 79) manifested moderate yeast inhibition through 24 hours. Depending upon the specific end-use application contemplated, it is evident that routine preliminary evaluations should be conducted before formulating with various additives to ascertain their impact on the inventive systems of the present invention.
EXAMPLES 80-95
Bioactives Synergy
A series of experiments were conducted in which possible synergies were evaluated between the inventive compositions and other bioactive materials as well as between such other bioactive materials including a fungicide, an antimicrobial agent and a disinfectant. The inventive bioactive system employed in this set of experiments (MI3) was a 4% aqueous citric acid solution containing 50 ppm silver, 50 ppm copper and 50 ppm zinc.
The fungicide evaluated was Mancozeb Flowable with Zinc from Bonide Products, Inc. of Oniskany, N.Y., USA, a commercial formulated fungicide containing 37% by wt mancozeb. Although the specific formulation of the Mancozeb product is proprietary, as a commercial formulation it would also contain certain surfactants for enabling its application to plants for efficacy. Mancozeb is an insoluble, dispersible powder that increases the turbidity of the liquids to which it is added. Nevertheless, in a separate evaluation, not reproduced here, it was found that Mancozeb was able to control or inhibit yeast growth at a concentration of about 1.23×10−3. The label indicates its use rate at 2.6×10−3.
The antimicrobial active evaluated was AgION AC10D, an antimicrobial zeolite additive available from AgION Technologies, Inc., of Wakefield, Mass., USA, which, as noted above, contains 6.0 wt % copper and 3.5 wt % silver. In a separate dilution evaluation, not reproduced here, it was found that an aqueous suspension of AC10D showed some yeast control or inhibition at a concentration of about 6.25×104.
Finally, the disinfectant evaluated was AgION SilverClene 24, a disinfectant material based on an aqueous solution of electrolytically generated silver citrate ( ̃30 ppm silver), also distributed by AgION Technologies, Inc. Although proprietary, this product and its manufacture is believed to be disclosed in Arata—U.S. Pat. No. 6,583,176, which is incorporated herein by reference in its entirety.
The aforementioned materials as well as various combinations thereof were evaluated to assess their efficacy in stopping or inhibiting the growth of yeast. The specific formulations tested and the yeast inhibition results attained therewith are presented in Tables 6 and 6A.










TABLE 6








AgION

Turbidity (NTU)


















Amt MI3
Mancozeb
AC10D
SilverClene 24

OD T
T
T
T



Example
(ml)
(wt %)
(wt %)
(ml)
Surfactant (wt %)
zero
(1 hour)
(18 hour)
(24 Hour)
pH




















80

9.40E−05



262
293
1023
1030
3.07


81
1
9.40E−05



276
276
309
522
2.91


82
2
9.40E−05



301
301
308
312
2.55


83
2
1.88E−04


0.05 NaLS/0.05 SLS
350
362
362
362



84
2
3.75E−04



656
640
1001
1170
2.4


85
1
9.40E−05


0.05 SLS
331
321
328
330
2.48


86
to pH 6
3.75E−04


0.05 NaLS/0.05 SLS
609
605
825
968
4.91


87

1.88E−04
7.81E−05

0.05 NaLS
410
385
443
511



88
2
1.88E−04
7.81E−05

0.05 NaLS/0.05 SLS
521
435
435
440
2.68


89

9.40E−05

1

258
276
970
962
2.67


90

1.88E−04

2

365
364
782
1048



91


3.90E−05


128
151
862
800
3.23


92
2

3.90E−05

0.05 SLS
154
156
172
175
2.54


93
2

1.56E−04

0.05 NaLS/0.05 SLS
190
143
148
156
2.66


94
2



0.05 NaLS/0.05 SLS
157
67
189
195
2.51


95
Control




73
98
898
856
3.25



















TABLE 6A








AgION

Change in Turbidity (delta NTU)


















Amt MI3
Mancozeb
AC10D
SilverClene 24

1
18
1-18
24
1-24


Example
(ml)
(wt %)
(wt %)
(ml)
Surfactant (wt %)
hour
hour
hour
hour
hour




















80

9.40E−05



31
761
730
768
737


81
1
9.40E−05



0
33
33
246
246


82
2
9.40E−05



0
7
7
11
11


83
2
1.88E−04


0.05 NaLS/0.05 SLS
12
12
0
12
0


84
2
3.75E−04



−16
345
361
514
530


85
1
9.40E−05


0.05 SLS
−10
−3
7
−1
9


86
to pH 6
3.75E−04


0.05 NaLS/0.05 SLS
−4
216
220
359
363


87

1.88E−04
7.81E−05

0.05 NaLS
−25
33
58
101
126


88
2
1.88E−04
7.81E−05

0.05 NaLS/0.05 SLS
−86
−86
0
−81
5


89

9.40E−05

1

18
712
694
704
686


90

1.88E−04

2

−1
417
416
683
682


91


3.90E−05


23
734
711
672
649


92
2

3.90E−05

0.05 SLS
2
18
16
21
19


93
2

1.56E−04

0.05 NaLS/0.05 SLS
−47
−42
5
−34
13


94
2



0.05 NaLS/0.05 SLS
−90
32
122
38
128


95
Control




25
825
800
783
758









The results presented in Tables 6 and 6A demonstrate a marked synergy between the inventive compositions according the present invention and commercial fungicides and antimicrobial agents. Specifically, for example, a comparison of the results for Examples 80, 81 and 82 demonstrate that combining low amounts of the metal ions, citric acid and fungicide provided excellent antifungal performance. While it is noted that these formulations did not have additional surfactant, the commercial fungicide itself contained surfactants that worked in combination with the metal ions and citric acid to provide the benefits owing to that combination as now claimed. These results show that excellent antifungal activity, as measured by yeast growth inhibition, may be attained with less than 10% of the amount of fungicide needed to inhibit yeast growth by the simple addition of low levels of acid and metal ions. As seen from Examples 91, 92 and 93, a similar synergy is shown for the inventive compositions in combination with a conventional inorganic antimicrobial agent. Here too, less than 10% of that amount of the antimicrobial agent needed when used alone, provided good antimicrobial performance when in combination with low levels of bioactive composition according to the present invention. However, the substitution of the SilverClene 24 for the inventive composition of the present invention, Examples 89 and 90, provided no apparent benefit despite the relatively high silver content.
Finally, in Example 86, ammonia was added to a portion of the MI3 solution until the solution reached a pH of 6. 2 ml of this buffered solution was then employed in the experiment. This example indicates the importance of the low pH of the compositions according to the present invention in order to provide desirable performance.
EXAMPLES 96-107
Immunox Synergy
A similar study was conducted to assess the synergy between the bioactive compositions according to the present invention and a second fungicide, Immunox, a commercial fungicide containing 1.55% myclobutanil, available from Spectrum Brands Division of United Industries of Madison, Wis., USA. As a commercial formulation, this too is expected to have some surfactants content. The bioactive composition employed in this experiment was the concentrated bioactive system (MI2) produced in Examples 72-79 above. The specific dilutions of each and the results attained thereby are presented in Table 7.












TABLE 7








Dilution Ratio

T1.5

Delta














Example
Immunox
MI2
T zero
OD
T18
T68 OD
68

















96

1:80 
150
152

832
682


97

1:200
106
112

980
874


98
1:64 

97
107
1043




99
1:128

111
119
1126




100
1:256

84
131

1170
1086


101
1:512

81
140

1240
1159


102
1:256
1:80 
138
141

268
130


103
1:256
1:200
102
114

1037
935


104
1:512
1:80 
138
140

292
154


105
1:512
1:200
97
110

1031
934


106
Control 1

86
175

754
668


107
Control 2

87
176

1180
1093









As indicated in Table 7, none of the test vials containing the low levels of each of the bioactive compositions or the Immunox dilution provided antifungal activity through the full 96 hour period tested. Furthermore, neither the 1:128 dilution (Example 99) nor the 1:64 dilution (Example 98) of Immunox provided any measure of efficacy, even in the shorter test period of 18 hours, despite the fact that the manufacturer generally recommends a dilution of 1:64. Similarly, Examples 103 and 105 having a 1:200 dilution of the bioactive composition ( ̃1 ppm of each metal, 0.08% citric acid, 0.00125 NaLS and 0.0016 SLS) in combination with the two dilutions of the Immunox failed to demonstrate bioefficacy whereas combinations of both dilutions of the Immunox with a somewhat higher level, 1:80 dilution, of the bioactive composition ( ̃2.5 ppm of each metal, 0.2% citric acid, 0.003 NaLS and 0.004 SLS) demonstrated bioefficacy. This demonstrates a synergy between the two compositions as the 1:80 dilution by itself failed to show bioefficacy over the full period tested.
EXAMPLES 108-126
Metal Sources
A series of experiments were conducted using different metal salts as the metal ion sources. Here, sufficient amounts of silver nitrate, copper sulfate and zinc oxide were added to a 5% aqueous citric acid solution to provide 31.75 ppm silver, 12.5 ppm copper and 40.17 ppm zinc. Different quantities of this stock concentrate solution (MI4) were added to the test vials to assess efficacy. The specific formulations, including the resultant ppm of each metal in the text vial, as well as the results thereof in inhibiting yeast growth were as presented in Tables 8 and 8A.
The results shown in Tables 8 and 8A demonstrate that the selection of the metal ion source is not critical so long as it is readily soluble and is soluble to the extent needed to provide the desired level of metal ion concentration in the solution. Furthermore, the results demonstrate the bioefficacy even at extremely low metal and acid contents. Although, the efficacy is relatively short lived at the lower concentrations, long-term bioefficacy is found with only minor adjustments in the relative concentration of the necessary components. Furthermore, depending upon the ultimate end-use application, such short term antifungal efficacy may be sufficient; thus, enabling one to minimize any environmental contamination from the general application of these materials.
The results also suggest that sodium lauryl sulfate may be ineffective on its own in promoting the bioefficacy of the bioactive compositions of the present invention. Nevertheless, its presence may be desirable where the efficacious surfactant is not readily soluble in the aqueous system. On the other hand, its presence or the presence of like surfactants may not be important where the intent is to produce non-aqueous systems. For example, systems to be applied as an emulsion in water or as an oil that will spread on an aqueous medium to which it is applied, e.g., a rice paddy, may look to surfactants that are less hydrophilic and more lipophilic.
EXAMPLES 127-143
Lactic Acid
A series of experiments was conducted similar to the previous with the exception that lactic acid was substituted for citric acid. Hence, the bioactive composition (MI5) comprised sufficient amounts of silver nitrate, copper sulfate and zinc oxide dissolved in a 5% aqueous lactic acid solution to provide 31.75 ppm silver, 12.5 ppm copper and 40.17 ppm zinc. The specific formulations tested and the results attained therewith were as presented in Tables 9 and 9A.











TABLE 8










Surfactant




Volume MI4
Metals Concentration
(w/w) %
Turbidity (NTU)




















Example
added
ppm Ag
ppm Cu
ppm Zn
NaLS
SDS
T zero
T2
T 18
T26
T44
T48
T68























108
0.5
0.79
0.31
1.00


81
129
950
1046
1046
1046
1054


109
1
1.59
0.63
2.01


85
136
950
997
1055
990
1023


110
2
3.18
1.25
4.02


112
158
916
930
960
930
970


111
3
4.76
1.88
6.03


126
158
760
799
810
830
844


112
0.5
0.79
0.31
1.00
0.005

140
143
179
307
919
936
980


113
1
1.59
0.63
2.01
0.005

140
137
143
152
279
306
468


114
2
3.18
1.25
4.02
0.005

180
174
174
177
244
252
282


115
3
4.76
1.88
6.03
0.005

187
185
184
184
184
184
272


116
0.5
0.79
0.31
1.00

0.005
83
132
948
1054
1066
1078
1097


117
1
1.59
0.63
2.01

0.005
97
136
911
1003
1100
1060
1075


118
2
3.18
1.25
4.02

0.005
116
147
746
907
970
1001
1006


119
3
4.76
1.88
6.03

0.005
124
156
504
701
840
868
916


120
0.5
0.79
0.31
1.00
0.0025
0.0025
140
140
250
640
1065
1088
1133


121
1
1.59
0.63
2.01
0.0025
0.0025
149
149
160
256
930
901
1014


122
2
3.18
1.25
4.02
0.0025
0.0025
164
177
174
174
291
459
804


123
3
4.76
1.88
6.03
0.0025
0.0025
176
179
177
181
320
445
736


124
2
3.18
1.25
4.02
0.01

162
162
162
163
163
164
164


125
0.86
1.37
0.54
1.73
0.01

150
140
140
140
186
208
254


126






78
113
877
866
878
865
898




















TABLE 8A









Metals





Volume
Concentration
Surfactant
Change in Turbidity (delta NTU)



















MI4
ppm
ppm
ppm
(w/w) %
Delta
DT18 −
DT26 −
DT44 −
DT48 −
DT68 −



















Example
added
Ag
Cu
Zn
NaLS
SDS
T2 − T0
T0
T0
T0
T0
T0






















108
0.5
0.79
0.31
1.00


48
869
965
965
965
973


109
1
1.59
0.63
2.01


51
865
912
970
905
938


110
2
3.18
1.25
4.02


48
804
818
848
818
858


111
3
4.76
1.88
6.03


32
624
673
684
704
718


112
0.5
0.79
0.31
1.00
0.005

3
39
167
779
796
840


113
1
1.59
0.63
2.01
0.005

−3
3
12
139
166
328


114
2
3.18
1.25
4.02
0.005

−8
−6
−3
64
72
102


115
3
4.76
1.88
6.03
0.005

−2
−3
−3
−3
−3
85


116
0.5
0.79
0.31
1.00

0.005
49
865
971
983
995
1014


117
1
1.59
0.63
2.01

0.005
39
814
906
1003
963
978


118
2
3.18
1.25
4.02

0.005
31
630
791
854
885
890


119
3
4.76
1.88
6.03

0.005
32
380
577
716
744
792


120
0.5
0.79
0.31
1.00
0.0025
0.0025
0
110
500
925
948
993


121
1
1.59
0.63
2.01
0.0025
0.0025
0
11
107
781
752
865


122
2
3.18
1.25
4.02
0.0025
0.0025
13
10
10
127
295
640


123
3
4.76
1.88
6.03
0.0025
0.0025
3
1
5
144
269
560


124
2
3.18
1.25
4.02
0.01

0
0
1
1
2
2


125
0.86
1.37
0.54
1.73
0.01

−10
−10
−10
36
58
104


126






35
799
788
800
787
820




















TABLE 9









Metals
Surfactant




Volume MI5
Concentration
(w/w) %
Turbidity (NTU)


















Example
added
ppm Ag
ppm Cu
ppm Zn
NaLS
SDS
T zero
T1
T18
T24
T44





















127
0.5
0.79
0.31
1.00


107
130
1000
1111
1001


128
1
1.59
0.63
2.01


109
130
1006
1021
1016


129
2
3.18
1.25
4.02


148
154
970
995
1014


130
3
4.76
1.88
6.03


178
202
914
925
990


131
0.5
0.79
0.31
1.00
0.005

134
170
300
454
923


132
1
1.59
0.63
2.01
0.005

153
169
200
227
292


133
2
3.18
1.25
4.02
0.005

218
217
207
204
228


134
3
4.76
1.88
6.03
0.005

222
223
222
215
227


135
0.5
0.79
0.31
1.00

0.005
120
145
1074
1111
1079


136
1
1.59
0.63
2.01

0.005
140
156
1050
1092
1110


137
2
3.18
1.25
4.02

0.005
179
193
945
1031
1080


138
3
4.76
1.88
6.03

0.005
223
239
690
977
1180


139
0.5
0.79
0.31
1.00
0.0025
0.0025
143
151
884
968
1170


140
1
1.59
0.63
2.01
0.0025
0.0025
175
175
237
330
1110


141
2
3.18
1.25
4.02
0.0025
0.0025
210
214
207
223
730


142
3
4.76
1.88
6.03
0.0025
0.0025
240
240
228
228
475


143
control





100
139
1175
1163
1170




















TABLE 9A









Metals
Surfactant




Volume MI5
Concentration
(w/w) %
Change in Turbidity (delta NTU)

















Example
added
ppm Ag
ppm Cu
ppm Zn
NaLS
SDS
DT1 − T0
DT18 − T10
DT24 − T0
DT44 − T0




















127
0.5
0.79
0.31
1.00


23
893
1004
894


128
1
1.59
0.63
2.01


21
897
912
907


129
2
3.18
1.25
4.02


8
822
847
866


130
3
4.76
1.88
6.03


24
736
747
812


131
0.5
0.79
0.31
1.00
0.005

36
166
320
789


132
1
1.59
0.63
2.01
0.005

16
47
74
139


133
2
3.18
1.25
4.02
0.005

−1
−11
−14
10


134
3
4.76
1.88
6.03
0.005

1
0
−7
5


135
0.5
0.79
0.31
1.00

0.005
25
954
991
959


136
1
1.59
0.63
2.01

0.005
16
910
952
970


137
2
3.18
1.25
4.02

0.005
14
766
852
901


138
3
4.76
1.88
6.03

0.005
16
467
754
957


139
0.5
0.79
0.31
1.00
0.0025
0.0025
8
741
825
1027


140
1
1.59
0.63
2.01
0.0025
0.0025
0
62
155
935


141
2
3.18
1.25
4.02
0.0025
0.0025
4
−3
13
520


142
3
4.76
1.88
6.03
0.0025
0.0025
0
−12
−12
235


143
control





39
1075
1063
1070









The results as shown in Tables 9 and 9A, mimic those found in the previous set of experiments indicating that the invention is translatable to acids of similar characteristics.
EXAMPLES 144-156
Phosphoric Acid
Two stock solutions were prepared for evaluation wherein the acid employed was phosphoric acid. In the first, silver citrate, copper citrate and zinc citrate were added to a 16% aqueous phosphoric acid solution to provide 200 ppm of each metal. A second stock solution was prepared using silver nitrate, copper sulfate and zinc oxide, again in the 16% phosphoric acid solution to provide 200 ppm of each metal. Both composition further contained 0.32% surfactant, either as an individual surfactant or as a 50:50 mix. The specific formulations and the results of their efficacy in controlling yeast growth were as presented in Tables 10 and 10A.
The results as shown in Tables 10 and 10A suggest that the surfactant may not be critical in those compositions wherein the excess acid is a strong to moderate acid, such as phosphoric acid.
EXAMPLES 157-166
Nitric Acid
To further demonstrate the breadth of the bioactive compositions, a relatively strong mineral acid, nitric acid, was employed as the acid component. A stock solution was prepared by combining 78.7 mg sliver nitrate, 62.2 mg zinc oxide and 200 mg copper sulfate with 20 ml of purified water and 1.5 g concentrated nitric acid (68%) under constant agitation. Once the solids were dissolved, additional purified water was added to make up a 250 volume. As prepared, this mixture contained approximately 200 ppm of each metal, as calculated. The pH was measured and found to be 1.66. The mixture was then divided into three aliquots of approximately equal volume. One aliquot was set aside and the other two were subjected to pH adjustment with ammonia hydroxide. The amount of ammonia hydroxide was added was that necessary to bring the pH of the first aliquot up to 2.55 and the second aliquot up to 3.63.
Each solution was then evaluated, with and without surfactants, to assess their bioefficacy in inhibiting the growth of yeast. The amount of each of the three aliquots added to the 20 ml vial of the yeast suspension is set forth in Table 11 together with the amount of surfactant added, where indicated. The surfactant employed was a 50:50 mix of sodium lauryl sulfate and sodium lauroyl sarcosinate. The specific formulations tested and the results thereof are presented in Table 11. As can be seen from Table 11, the combination of metal and acid did not provide any inhibition at the levels tested. However, when the surfactant was added, bioefficacy was manifested even at the lower metal/acid concentration.








TABLE 10








Turbidity (NTU


















Example
Metal source
Metal (ppm)
Surfactants (w/w)
T zero
T1 hour
T18
T24
T42
T48
T72
T96





















144
Citrate salts*
2.5

123
134
300
400
1046
1094
1146
1106


145
Citrate salts*
5

199
180
166
166
160
163
162
154


146
Citrate salts*
10

211
193
176
176
172
177
172
169


147
AgNO3, CuSO4, ZnO
2.5

168
166
179
179
172
174
778
1162


148
AgNO3, CuSO4, ZnO
5

209
193
180
180
175
174
170
168


149
AgNO3, CuSO4, ZnO
10

228
219
197
197
196
204
199
194


150
Citrate salts*
5
0.05 SLS
226
218
200
200
193
203
192
186


151
Citrate salts*
5
0.05 NaLS
258
254
216
216
200
205
197
185


152
Citrate salts*
5
0.05 SLS/0.05 NaLS
253
237
200
200
204
208
201
188


153
AgNO3, CuSO4, ZnO
5
0.05 SLS
285
263
229
229
223
229
214
206


154
AgNO3, CuSO4, ZnO
5
0.05 NaLS
280
273
226
222
216
213
208
184


155
AgNO3, CuSO4, ZnO
5
0.05 SLS/0.05 NaLS
283
272
250
247
232
238
232
215


156
Control


52
53
437
599
938
913
877
886





*Ag citate, Cu citrate and Zn citrate, each at level designated
















TABLE 10A








Metal

Change in Turbidity (delta NTU)

















Example
Metal source
(ppm)
Surfactants (w/w)
T1 − T0
T18 − T1
T24 − T1
T42 − T1
T48 − T1
T72 − T1
T96 − T1




















144
Citrate salts*
2.5

11
166
266
912
960
1012
972


145
Citrate salts*
5

−19
−14
−14
−20
−17
−18
−26


146
Citrate salts*
10

−18
−17
−17
−21
−16
−21
−24


147
AgNO3, CuSO4, ZnO
2.5

−2
13
13
6
8
612
996


148
AgNO3, CuSO4, ZnO
5

−16
−13
−13
−18
−19
−23
−25


149
AgNO3, CuSO4, ZnO
10

−9
−22
−22
−23
−15
−20
−25


150
Citrate salts*
5
0.05 SLS
−8
−18
−18
−25
−15
−26
−32


151
Citrate salts*
5
0.05 NaLS
−4
−38
−38
−54
−49
−57
−69


152
Citrate salts*
5
0.05 SLS/0.05 NaLS
−16
−37
−37
−33
−29
−36
−49


153
AgNO3, CuSO4, ZnO
5
0.05 SLS
−22
−34
−34
−40
−34
−49
−57


154
AgNO3, CuSO4, ZnO
5
0.05 NaLS
−7
−47
−51
−57
−60
−65
−89


155
AgNO3, CuSO4, ZnO
5
0.05 SLS/0.05 NaLS
−11
−22
−25
−40
−34
−40
−57


156
Control


1
384
546
885
860
824
833
















TABLE 11







Nitric Acid













Vol. MI6
Metals
Surfactant

Turbidity/Change in Turbidity
















Example
Added
(ppm)
(w/w) %
pH
T0
T18
T18 − T0
T42
T42 − T0



















157
0.5
5

1.66
69
1243
1174
1133
1064


158
0.5
5

2.55
67
1245
1178
1133
1066


159
0.5
5

3.63
69
1243
1174
1150
1081


160
1
10

1.66
65
976
911
1162
1097


161
1
10

2.55
66
1012
946
1186
1120


162
1
10

3.63
67
1036
969
1166
1099


163
0.5
5
0.05
1.66
61
55
−6
58
−3


164
0.5
5
0.05
2.55
62
53
−9
55
−7


165
0.5
5
0.05
3.63
60
57
−3
52
−8


166
0



67
1255
1188
1212
1145









EXAMPLES 167-222
Surfactant Evaluation
A series of experiments were conducted to screen various surfactants for efficacy in accordance with the present invention. The surfactants were evaluated as a neat additive (0 ppm metals) or in combination with either 1 ml or 2 ml of a 4% citric acid solution containing 50 ppm each of copper, silver and zinc. With the addition of 1 ml of the citric acid solution, the test vial of the yeast suspension will have about 0.2% citric acid and about 2.5 ppm of each metal. With the addition of 2 ml of the citric acid solution, the acid is approximately 0.4% and the metals are each present at about 5 ppm in the test vials. Each surfactant was evaluated at a concentration of approximately 0.05 wt %. Controls were also evaluated with and without the metals.
The specific surfactants evaluated as well as the formulations of each test composition together with the results thereof are set forth in Table 12. As seen in Table 12, the benefits of the present invention are realized with a broad array of surfactant materials. Especially preferred surfactants are those that are free or substantially free of repeat ethylene oxide units and/or have moderate to lower molecular weights. Despite the foregoing, it is noted that good results were attained with the Pluronic L62, a polyethylene oxide containing surfactant, when used in combination with the lower level of acid and metals. It is thought that the higher acid level may have affected the stability of this material, and possibly like materials.





























Metal





T18 −
T48 −
T72 −
T96 −


Surfactants
Surfactant Chemistry
Source
Type
ppm
T0
T18
T48
T72
T96
T0
T0
T0
T0





Pluronic L62
E0-PO Block
BASF
Nonionic
0
47
1088
1113
1142
1156
1041
1066
1095
1109



copolymer


2.5
343
376
362
364
340
33
19
2
−24






5
118
1127
1138
1175
1146
1009
1020
1057
1028


Hampopsyl L95
Na N-lauroyl
Hampshire
Anionic
0
47
42
390
884
878
−5
343
837
831



Sarcosinate
Chemical

2.5
70
909
999
1037
983
839
929
967
913






5
407
444
442
440
440
37
35
33
33


Sodium Lauryl
Sodium Lauryl Sulfate
VWR Scientific
Anionic
0
48
495
658
642
639
447
610
594
591


Sulfate



2.5
88
90
88
88
87
2
0
0
−1






5
231
244
233
238
232
13
2
7
1


Witco
Sodium Laurylether
Witco Chemical
Anionic
0
48
1060
1021
957
923
1012
973
909
875



Sulfate (2 mole EO)


2.5
73
819
1415
1436
1447
746
1342
1363
1374






5
140
143
446
870
915
3
306
730
775


Jeenteric
Cocamidopropyl
Jeen
Amphoteric
0
48
645
657
882
462
597
609
834
414


CAPB LC
betaine
International

2.5
93
90
91
90
88
−3
−2
−3
−5




Corp

5
204
204
202
202
202
0
−2
−2
−2


Manckinate
Dilauryl
Mackintire
amphoteric
0
95
1020
866
817
788
925
771
722
693


LO100 DLSS
sulfosuccinate
Chemical

2.5
118
97
106
1165
1317
−21
−12
1047
1199






5
251
239
232
224
215
−12
−19
−27
−36


Ammonyx LO
Lauryl Dimethyamine
Stepan Chemical
Nonionic
0
44
28
35
45
28
−16
−9
1
−16



Oxide


2.5
972
390
118
115
105
−582
−854
−857
−867






5
652
314
252
227
180
−338
−400
−425
−472


Hamposyl C30
Na N-cocoyl
Hampshire
Anionic
0
44
207
1043
1041
1037
163
999
997
993



Sarcosinate
Chemical

2.5
699
677
657
673
1115
−22
−42
−26
416






5
510
554
576
589
593
44
66
79
83


Hamposyl M30
Na N-myristoyl
Hampshire
Anionic
0
46
28
152
1205
1184
−18
106
1159
1138




Chemical

2.5
588
564
1372
1385
1389
−24
784
797
801



Sarcosinate


5
583
586
1299
1382
1383
3
716
799
800


Hampshire TL
TEA lauroyl
Hampshire
Anionic
0
66
946
977
927
905
880
911
861
839


Glutamate
Glutamate
Chemical

2.5
182
410
1143
1189
1178
228
961
1007
996






5
218
618
1104
1129
1162
400
886
911
944


Tergitol 15S3
Secondary Alcohol
Dow Chemical
Nonionic
0
188
1140
1178
969
880
952
990
781
692



Ethoxylate


2.5
180
340
1247
1227
1134
160
1067
1047
954






5
317
818
1350
1297
1289
501
1033
980
972


Tergitol 15S7
Secondary Alcohol
Dow Chemical
Nonionic
0
48
865
1077
766
577
817
1029
718
529



Ethoxylate


2.5
91
117
1152
1087
917
26
1061
996
826






5
197
408
1291
1224
1217
211
1094
1027
1020


Tergitol TMN6
Branched Secondary
Dow Chemical
Nonionic
0
50
940
1128
784
614
890
1078
734
564



Alcohol Ethoxylate


2.5
106
132
1184
1140
1048
26
1078
1034
942






5
215
480
1300
1275
1266
265
1085
1060
1051


Tergitol TMN3
Branched Secondary
Dow Chemical
Nonionic
0
49
314
1015
700
541
265
966
651
492



Alcohol Ethoxylate


2.5
92
94
1054
1014
876
2
962
922
784






5
189
247
1100
1128
1128
58
911
939
939


Sulfonic TDA3B
C1-C14 Ethoxylated
Huntsman
Nonionic
0
206
1163
1183
948
809
957
977
742
603



Alcohol
Chemical

2.5
260
372
1296
1248
1192
112
1036
988
932






5
359
725
1369
1366
1319
366
1010
1007
960


Tween 20
polyoxyethylene (20)

Nonionic
0
57
1077
1118
1087
730
1020
1061
1030
673



sorbitan monolaurate


2.5
92
932
1116
867
719
840
1024
775
627






5
169
1080
1144
1105
1048
911
975
936
879


Plantaren 2000
Alkyl polyglycoside
Cognis
Nonionic
0
56
346
906
782
642
290
850
726
586






2.5
102
410
660
1104
1323
308
558
1002
1221






5
229
235
232
232
237
6
3
3
8


Control



0
58
1171
1152
1168
1177
1113
1094
1110
1119


Control (2.5 ppm)



0
94
968
1073
1180
1041
874
979
1086
947


Control (5 ppm)



0
132
1196
1185
1228
1233
1064
1053
1096
1101


Metals Control



2.5
93
1001
1080
1128
962
908
987
1035
869


Metals Control



5
152
1160
1186
1228
1193
1008
1034
1076
1041









EXAMPLES 223-236
Strobilurin Comparison
A series of experiments were conducted in order to evaluate the comparative performance of the bioactive compositions of the present invention and several commercial strobilurin based fungicides. Two bioactive formulations were used. The first, MI2, comprised a 16% aqueous citric acid solution having dissolved therein silver citrate, copper citrate and zinc citrate, each being added in an amount to provide 200 ppm of each metal, together with 0.25% sodium Lauroyl sarcosinate and 0.32% sodium lauryl sulfate, as noted above. The second, MI7, comprised a 160:1 dilution of a 16% aqueous phosphoric acid solution having dissolved therein silver citrate, copper citrate and zinc citrate, each being added in an amount to provide 200 ppm of each metal in the phosphoric acid solution. Each fungicide was evaluated at different levels. The specific formulations tested and the results attained therewith are presented in Tables 13 and 13A.
As seen in Tables 13 and 13A, the bioactive compositions of the present invention provided marked inhibition of yeast growth, even at the lower concentrations,  ̃5 ppm of each metal ion. On the other hand, all but two of the strobilurin based fungicide formulations tested failed to demonstrate any significant bioefficacy against yeast over the time period tested. The two formulations that provided good inhibition were at comparatively high loadings.
EXAMPLES 237-250
Strobilurin Synergy
In light of the foregoing poor performance of the strobilurins generally, a series of experiments were conducted in order to evaluate the potential synergy between the bioactive compositions of the present invention and the foregoing commercial strobilurin based fungicides. The compositions employed were the same as used in the previous set of examples. The specific formulations tested and the results attained therewith are presented in Tables 14 and 14A.








TABLE 13








Turbidity (NTU)














Example
Fungicide
vol. Added
T0
T1
T18
T26
T50

















223
Quadrisa
1
384
393
1066
1139
1134


224

2
767
772
1264
1311
1315


225

5
1332
1332
1364
1377
1376


226
Flintb
1
418
424
1115
1208
1234


227

2
718
708
1141
1299
1327


228

5
1210
1210
1270
1265
1245


229
Headlinec
1
232
225
961
1114
1137


230

2
387
391
1066
1134
1199


231

5
717
747
1178
1222
1241


232
MI2
0.5
128
129
154
177
174


233
MI2
1
414
384
366
366
352


234
MI7
0.5
249
244
248
248
242


235
MI7
1
311
302
283
283
277


236
Control

67
68
793
871
904





aQuadris fungicide from Syngenta Crop Protections, Inc. of Greensboro, NC, USA


bFlint fungicide from Bayer CropScience LP of Research Triangle Park, NC, USA


cHeadline from BASF Corporation of Research Triangle Park, NC, USA














TABLE 13A








Change in Turbidity (delta NTU)












Example
Fungicide
vol. Added
T18 − T1
T26 − T1
T50 − T1















223
Quadrisa
1
673
746
741


224

2
492
539
543


225

5
32
45
44


226
Flintb
1
691
784
810


227

2
433
591
619


228

5
60
55
35


229
Headlinec
1
736
889
912


230

2
675
743
808


231

5
431
475
494


232
MI2
0.5
25
48
45


233
MI2
1
−18
−18
−32


234
MI7
0.5
4
4
−2


235
MI7
1
−19
−19
−25


236
Control

725
803
836





aQuadris fungicide from Syngenta Crop Protections, Inc. of Greensboro, NC, USA


bFlint fungicide from Bayer CropScience LP of Research Triangle Park, NC, USA


cHeadline from BASF Corporation of Research Triangle Park, NC, USA














TABLE 14








Turbidity (NTU)
















Example
Bioactive
Vol. Added
Fungicidea
Vol. Added
T0
T1
T18
T24
T96



















237
MI2
0.25
Q
1
552
554
544
670
1315


238
MI2
0.25
Q
2
896
894
868
891
1470


239
MI2
0.5
Q
1
588
578
564
564
608


240
MI2
0.25
F
1
578
599
568
568
1320


241
MI2
0.25
F
2
900
900
886
886
1330


242
MI2
0.25
H
1
436
433
454
454
1312


243
MI2
0.25
H
2
611
637
667
632
1302


244
MI7
0.25
Q
1
558
574
640
668
1273


245
MI7
0.25
F
1
517
560
990
1197
1396


246
MI7
0.25
H
1
465
476
605
587
1290


247
Control

—

93
101
901
986
1075


248
MI2
0.5


499
440
390
390
373


249
MI2
0.25


182
179
175
176
1122


250
MI2
0.5


262
260
260
275
275





aQ—Quadris fungicide from Syngenta Crop Protections, Inc. of Greensboro, NC, USA; F—Flint fungicide from Bayer CropScience LP of Research Triangle Park, NC, USA; and H—Headline from BASF Corporation of Research Triangle Park, NC, USA














TABLE 14A








Change in Turbidity (delta NTU)














Example
Bioactive
Vol. Added
Fungicidea
Vol. Added
T18 − T1
T24 − T1
T96 − T1

















237
MI2
0.25
Q
1
−10
116
761


238
MI2
0.25
Q
2
−26
−3
576


239
MI2
0.5
Q
1
−14
−14
30


240
MI2
0.25
F
1
−31
−31
721


241
MI2
0.25
F
2
−14
−14
430


242
MI2
0.25
H
1
21
21
879


243
MI2
0.25
H
2
30
−5
665


244
MI7
0.25
Q
1
66
94
699


245
MI7
0.25
F
1
430
637
836


246
MI7
0.25
H
1
129
111
814


247
Control

—

800
885
974


248
MI2
0.5


−50
−50
−67


249
MI2
0.25


−4
−3
943


250
MI2
0.5


0
15
15





aQ—Quadris fungicide from Syngenta Crop Protections, Inc. of Greensboro, NC, USA; F—Flint fungicide from Bayer CropScience LP of Research Triangle Park, NC, USA; and H—Headline from BASF Corporation of Research Triangle Park, NC, USA






As seen in Tables 14 and 14A, the combination of the bioactive compositions of the present invention with the strobilurin products produced a synergy whereby even the lowest levels of the strobilurin products tested produced a significant inhibition in yeast growth, even though these products appear to increase yeast growth when used alone, as shown in the Tables 13 and 13A.
EXAMPLES 251-259
Copper/Zinc Study
A series of experiments were conducted to demonstrate the bioefficacy of binary metal systems as compared to the ternary system used in most other examples. Here a solution of MI2 was compared to a similar composition containing 300 ppm of copper and 300 ppm of zinc (i.e., a 16% aqueous citric acid solution having dissolved therein copper citrate and zinc citrate, each being added in an amount to provide 300 ppm of each metal, together with 0.25% sodium Lauroyl sarcosinate and 0.32% sodium lauryl sulfate). The two bioactive compositions were evaluated at different loadings to assess their bioefficacy. The specific formulations tested and the results attained therewith are presented in Tables 15 and 15A.
As seen in Tables 15 and 15A, both the binary (copper/zinc—Cu/Zn) and the MI2 ternary silver/copper/zinc antimicrobial bioactive compositions demonstrated comparable bioefficacy in inhibiting the growth of yeast.









TABLE 15








Composition (gm)















Example
Cu/Zn
MI2
T0
T1
T18
T24
T46

















251
1

776
586
468
463
436


252
0.5

292
269
250
250
245


253
0.2

147
162
772
1055
1075


254
0.1

93
125
1076
1070
1036


255
Control

66
127
1020
1012
1137


256

1
830
633
547
522
500


257

0.5
335
320
292
302
284


258

0.2
152
178
512
1064
1098


259

0.1
90
136
1083
1087
1067


















TABLE 15A








Composition (gm)















Cu/Zn
MI2
T1 − T0
T18 − T0
T24 − T0
T46 − T0
















251
1

−190
−118
−5
−27


252
0.5

−23
−19
0
−5


253
0.2

15
610
283
20


254
0.1

32
951
−6
−34


255
Control

61
893
−8
125


256

1
−197
−86
−25
−22


257

0.5
−15
−28
10
−18


258

0.2
26
334
552
34


259

0.1
46
947
4
−20









EXAMPLES 260-269
Mancozeb Synergy
A further series of experiments were conducted to assess the bioefficacy, especially the synergy, of the bioactive agrichemical composition containing Mancozeb (an ethylene bisdithiocarbamate) and the MI2 bioactive acid solution (MI2). The specific formulations tested and the results attained therewith are presented in Table 16 and 16A.









TABLE 16








Composition (gm)















Example
Mancozeb
MI2
T0
T2
T18
T24
T44

















260
0.5

934
976
1220
1095
1091


261
0.4

780
859
1021
982
1052


262
0.3

624
717
1209
1067
1113


263
0.2

392
489
1035
933
1073


264

0.2
57
55
54
72
756


265
0.5
0.2
930
897
864
839
788


266
0.4
0.2
727
709
684
664
591


267
0.3
0.2
537
555
535
509
460


268
0.2
0.2
370
369
370
343
331


269
Control

23
106
935
824
917


















TABLE 16A








Composition



Exam-
(gm)














ple
Mancozeb
MI2
T2 − T0
T18 − T0
T24 − T0
T44 − T0
















260
0.5

42
286
161
157


261
0.4

79
241
202
272


262
0.3

93
585
443
489


263
0.2

97
643
541
681


264

0.2
−2
−3
15
699


265
0.5
0.2
−33
−66
−91
−142


266
0.4
0.2
−18
−43
−63
−136


267
0.3
0.2
18
−2
−28
−77


268
0.2
0.2
−1
0
−27
−39


269
Control

83
912
801
894









As seen in Tables 16 and 16A, the mancozeb by itself was ineffective at all levels tested. The bioactive acid solution by itself provided modest bioefficacy, in spite of the very low level of antimicrobial metal ions; however, the suitable bioefficacy appeared to have been lost after 44 hours. In sharp contrast, the combination of the two, at all levels of the mancozeb, demonstrated excellent bioefficacy, even after 44 hours.
EXAMPLES 270-293
Amine Oxide Surfactant Study
A series of experiments were conducted to demonstrate the bioefficacy of amine oxide surfactants, specifically, lauryl dimethyl amine oxide (LDAO), alone and in combination with sodium lauroyl sarcosinate (NaLS) and/or sodium lauryl sulfate (SLS). In this instance a very dilute antimicrobial metal-acid solution was employed: 0.08% citric acid and 1 ppm each of silver, copper and zinc. The surfactants were employed at different levels to assess the lowest concentration at which synergy is realized. The specific formulations tested and the results attained therewith are presented in Table 17.
As seen in Table 17, even at such low concentration of acid and metal, the addition of only 0.0025% lauryl dimethyl amine oxide surfactant showed bioefficacy, with modest bioefficacy at the 0.00125% level with sodium lauroyl sarcosinate or the combination of sodium lauroyl sarcosinate and/or sodium lauryl sulfate. At 0.0025% lauryl dimethyl amine oxide, marked bioefficacy was found with addition of sodium lauroyl sarcosinate and superior bioefficacy found with addition of both sodium lauroyl sarcosinate and sodium lauryl sulfate.

















TABLE 17






LDAO
NaLS
SLS
AG, Cu, Zn








Example
(w/w)%
(w/w)%
(w/w)%
ppm
T zero
T1
T42
T66
T42 − T1
T66 − T1

























270
0.00025



132
219
1145
1133
926
914


271
0.00125



141
211
1120
1039
909
828


272
0.0025



161
196
862
814
666
618


273
0.00025


1
142
209
1108
1138
899
929


274
0.00125


1
144
208
1080
1076
872
868


275
0.0025


1
156
208
963
969
755
761


276




144
239
1232
1216
993
977


277
0.00025
0.00025


144
217
1084
1042
867
825


278
0.00125
0.00125


136
169
860
784
691
615


279
0.0025
0.0025


136
136
562
543
426
407


280
0.00025
0.00025

1
150
216
1032
1021
816
805


281
0.00125
0.00125

1
165
186
872
852
686
666


282
0.0025
0.0025

1
174
184
181
295
−3
111


283

0.00025


149
248
1138
1165
890
917


284

0.00125


142
202
1019
1018
817
816


285

0.0025


147
207
1034
1007
827
800


286



1
153
242
1167
1178
925
936


287




165
270
1223
1207
953
937


288
0.00025
0.00025
0.00025

178
272
1094
1006
822
734


289
0.00125
0.00125
0.00125

167
242
800
686
558
444


290
0.0025
0.0025
0.0025

224
212
605
550
393
338


291
0.00025
0.00025
0.00025
1
171
252
1039
1010
787
758


292
0.00125
0.00125
0.00125
1
260
258
862
872
604
614


293
0.0025
0.0025
0.0025
1
264
257
242
242
−15
−15









Antibacterial Study
EXAMPLES 294-325
A series of experiments were conducted to evaluate the performance of the individual components of the claimed bioactive compositions as well as various combinations thereof, including, the claimed compositions themselves, in suppressing the growth of various bacteria. Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) were selected as a test organisms as they are generally accepted in the industry as indicator organisms for a wide variety of bacteria. Two different test methodologies were evaluated, one testing the efficacy in a growth broth media and the other testing inhibition in plated growth media.
EXAMPLES 309-321
In the first set of experiments a growth medium was prepared by adding 10 grams of nutrient medium (Difco Sabouraud dextrose broth from BD of Franklin Lakes, N.J., USA) to 300 ml of distilled water. The 20 ml aliquots of the growth medium were dispensed into sterile into 40 ml borosilicate glass vials with Teflon lined caps (VWR International Cat. No. 15900-004). The vials were inoculated with the bacteria using a sterile loop and the vials then incubated at 37° C. A bioactive composition according to the invention was then added to certain vials, the bioactive composition was (MI2), as described above, comprising a 16% aqueous citric acid solution having dissolved therein silver citrate, copper citrate and zinc citrate, each added in an amount to provide 200 ppm of each metal, together with 0.25% sodium Lauroyl sarcosinate and 0.32% sodium lauryl sulfate. The turbidity of each mixture was then determined and the vial transferred to an incubator at 30° C. Turbidity measurements were performed as in the above cited yeast studies. Each vial was periodically removed from the incubator and the mixture in the vials assessed for turbidity. The specific formulations tested, the timing for each turbidity evaluation, and the results attained thereby were as set forth in Table 18.
As with the yeast study, the concentration of the metals refers the approximate amount of each metal, copper, silver and zinc. The concentrations do not account for the volume of MI2 added: thus, the concentrations presented are on the basis of a 20 ml total volume.
As seen in Table 18, there was short term increase in turbidity. Since it was not anticipated that any significant growth would have manifested in such a short period of time, it is believed that the initial increase in turbidity resulted from a denaturation of proteins in the broth and/or bacterial proteins. Regardless, the longer term results show excellent bacterial inhibition with the compositions according to the present invention.










TABLE 18








MI2

Time (hours)















Example
Bacterium
(ml)
Metals ppm
T0
T0.5
T18
T24
T96


















294
E. coli
0
0
15.3
16
119
136
264


295

0.5
5
131
135.3
165
162
162


296

1
10
445
454
481
480
480


297

2
20
1039
1080
1135
1140
1009


298
p. aeruginosa
0
0
35.8
37.8
158
383
436


299

0.5
5
197
207
250
262
261


300

1
10
705
735
782
808
807


301

2
20
1011
1057
1121
1159
1146


302
S. aureus
0
0
46
45
148
184
406


303

0.5
5
215
163
173
183
184


304

1
10
643
494
326
309
276


305

2
20
1203
1032
595
525
281









EXAMPLE 306
In this experiment, six 25 mm sterile coverslips were placed into separate 100×15 mm sterile Petri dishes and two of each inoculated with 100 μl of one of three TSB broths: each broth containing one of E. coli, P. aeruginosa and S. aureus that had been allowed to incubate for 48-54 hours. In order to afix the inoculum to the coverslips, the Petri dishes were placed on a low temperature hot plate for approximately 5 minutes. One of each of the inoculated Petri dishes was set aside as positive controls. The other was sprayed with 4 sprays of a 4:1 dilution of the bioactive compositions MI2. After 2-3 minutes the coverslips and liquid contents of each Petri dish was aseptically transferred into separate vials containing 20 ml of TSB and incubated at 37° C. for 24 hours. Negative controls were prepared by placing non-inoculated sterile coverslips into the 20 ml TSB and incubating as well. After 24 hours, no growth was observed with the negative controls or with those inoculated coverslips that had been sprayed with the bioactive composition of the present invention. Visual growth was observed in two of the positive controls (i.e., those vials containing the inoculated coverslips that had not been sprayed): the positive control for p. aeruginosa failed to show visual growth. It is believed that the failure of the later to show growth resulted from overheating the inoculum during the fixturing step.
EXAMPLE 307
In this experiment, two Trypticase soy agar (TSA) plates were inoculated with 500 μl of one of three TSB broths for a total of 6 inoculated plates: each broth contained one of E. coli, P. aeruginosa and S. aureus that had been allowed to incubate for 48-54 hours. The inoculum was evenly spread across the surface of the plate with a sterile loop. A 15 mm diameter disc of filter paper that had been dipped in a 4:1 dilution of the MI2 bioactive composition was placed in the center of one of each set of inoculated plates and all plates were placed in an incubator at 37° C. for 24 hours. Non-inoculated control plates were also placed in the incubator as well.
After 24 hours, visual growth was observed. No bacterial growth was seen in the non-inoculated plates. Growth was observed on all of the inoculated plates; however, in those plates wherein the treated filter paper had been placed, no growth was seen on or near the filter paper. Each treated filter paper disc manifested a clear zone of inhibition of bacterial growth.
EXAMPLE 308
In this experiment, two Trypticase soy agar (TSA) plates were innoculated with 500 μl of one of three TSB broths for a total of 6 inoculated plates: each broth contained one of E. coli, P. aeruginosa and S. aureus that had been allowed to incubate for 48-54 hours. The inoculum was evenly spread across the surface of the plate with a sterile loop. One of each inoculated plates was then sprayed, approximately 24 times, with the 4:1 dilution of the MI2 bioactive composition. The inoculated plates plus a set of plates non-inoculated control plates were placed in an incubator at 37° C. for 24 hours.
After 24 hours, visual growth was observed on inoculated, but untreated plates whereas no bacterial growth was seen in the non-inoculated plates or in those inoculated plates that had been sprayed with the diluted bioactive composition.
EXAMPLES 309
Bacterial MIC Study
A study was conducted to determine the minimum inhibitory concentration (MIC) of the MI2 acid solution, i.e., 200 ppm of each of silver, copper and zinc metal (see Examples 72-79). Three different bacteria were evaluated, Clavibacter michiganese, Pseudomonas syringae and Erwinia amylovora, each in a different growth medium appropriate for that bacteria, namely brain infusion agar/broth, nutrient agar/broth, and nutrient glucose agar/broth, respectively. In conducting the test, three sets of 10 test tubes were prepared, one set for each bacteria, and labeled 1 to 10. 0.5 ml of the appropriate broth was placed in each of test tubes 2 through 10. Then 0.5 ml of the MI2 solution was added to each of test tubes 1 and 2. 0.5 ml of the contents of test tube 2 was then transferred to test tube 3 and then 0.5 ml of test tube 3 to test tube 4 and so on to test tube 9. 0.5 ml or test tube 9 was discarded. A 0.5 ml suspension of each bacteria to be tested was then added to each of the ten tubes for that series and the tubes incubated for 24 hours at 26° C. Because the acid solution caused considerable cloudiness of the tubes to which it was added, macroscopic evaluation was not possible. Instead, each tube was subcultured onto corresponding agar plates. The observed growth was as indicated in Table 19 (a “+” indicates visual growth and a “−” no growth).








TABLE 19








Test Tube


















1
2
3
4
5
6
7
8
9
10





Metals
200
50
25
12.5
6.75
3.125
1.56
0.782
0.391
0.195


concentration* (ppm)












C. michiganese
−
−
−
−
−
−
+
+
+
+


P. syringae
−
−
−
−
−
+
+
+
+
+


E. amylovora
−
−
−
−
−
−
+
+
+
+





*concentration of each metal, the total metal content is 3 time the number presented.






Based on the results presented in Table 19, the MIC of MI2 is 3.125 ppm for C. michiganese and for E. amylovora and 6.75 ppm for P. syringae. The bioefficacy of such low levels are anticipated to show synergy when combined with conventional fungicides/bactericides for these target organisms
EXAMPLE 310
Alternaria Leaf Spot
To demonstrate the efficacy of the bioactive compositions on live plants, a comparative study was conducted comparing the efficacy of a bioactive composition according to the present invention to two commercial products, Eagle 40WP, a myclobutanil based (40 wt %) fungicide available from Dow AgroSciences LLC of Indianapolis, Ind., USA, and Scala SC, a pyrimethanil based (54.6 wt %) fungicide available from Bayer CropScience LP of Research Triangle Park, N.C., USA. Additional evaluations were conducted to assess the potential for synergy between the inventive bioactive compositions and Eagle 40WP. The bioactive composition according to the present invention comprised a 16% aqueous citric acid solution having dissolved therein silver citrate, copper citrate, and zinc citrate in an amount to provide 200 ppm of each metal in the solution, 0.25% sodium lauroyl sarcosinate and 0.32% sodium lauryl sulfate (MI6). This solution was diluted at rates of 40:1 and 20:1 for application to the plants thereby providing a solution containing  ̃5 ppm and  ̃10 ppm of each metal as sprayed.
Pittosporum tobira “Wheeleri” rooted cuttings were planted in standard 4 inch pots containing Sunshine Mix No. 1 and fertilized with 1⁄2 tsp. Osmocote Plus 15-9-12. The plants were placed in a heated greenhouse with poly and shade cloth covering the top and sides and flood irrigated as needed. After 44 days, the plants were treated with the various antifungal treatments—12 plants were treated with each treatment. Thereafter, the plants were placed in individual clear plastic bags (high humidity) in the greenhouse for the duration of the study. The plants were irrigated from below using an ebb and flood bench to assure no water application to their leaves during the trial. The plants were subsequently inoculated by spraying with a spore suspension of a culture of Alternaria piffospori mixed with sterilized water 4 days following the initial treatment. The treatments were reapplied 7 days and 17 days following inoculation. All treatments were applied by spray until the surfaces of the plant leaves were fully wetted (began to drip). Two sets of plants were used as positive and negative controls: the first set was treated with water only (Treatment A) and not inoculated. The second set was also treated with water only, but was also inoculated concurrent with the others. The specific formulations for each of the treatments were as set forth in Table 20.









TABLE 20





Treatment
Composition
Dilution







A
Water - noninoculated



B
Water - inoculated



C
MI6
6.25 ml/250 ml water


D
MI6
12.5 ml/250 ml water


E
MI6/Eagle 40WP
6.25 ml/250 ml water//




1.5 oz/100 gal water


F
MI6/Eagle 40WP
6.25/250 ml water//




3.0 oz/100 gal water


G
MI6/Eagle 40 WP
12.5 ml/250 ml water//




1.5 oz/100 gal water


H
Eagle 40 WP
1.5* oz/100 gal water


I
Eagle 40WP
3.0 oz/100 gal water


J
Scala
9.0* oz/100 gal water





*manufacturer recommended application rates






Six days following the second treatment, the plants were evaluated for Alternaria leaf spot by visual inspection. The results of the leaf spot evaluation were as presented in Table 21. As seen in Table 21, those plants treated with the lowest concentration of the bioactive composition (with  ̃5 ppm of each metal ion—Treatment C) still showed nearly a 50% drop in leaf spot formation. Doubling the bioactive composition ( ̃10 ppm of each metal ion—Treatment D) reduced leaf spot by over 75%. Somewhat similar results were found with the two dilutions of the commercial fungicide Eagle 40WP with the lower concentration (Treatment H) reducing leaf spot by about 30% while the higher concentration (Treatment I) reduced leaf spot by 80%. Combining the two provided marked improvement with, oddly enough, the combination of the two lowest concentrations providing nearly complete inhibition of leaf spot manifestation. The other commercial fungicide Scala SC provided no inhibition and, appeared to promote the manifestation of leaf spot.









TABLE 21







Treat-
Plant No.





















ment
1
2
3
4
5
6
7
8
9
10
11
12
Mean























A.
0
0
0
0
0
0
0
0
0
0
0
0
0.0


B.
4
5
0
15
35
20
40
15
10
25
30
20
18.2


C.
0
0
0
0
0
5
35
35
40
0
0
0
9.6


D.
0
1
0
0
5
0
0
0
0
5
10
30
4.2


E.
0
0
0
0
0
1
0
0
5
0
0
0
0.5


F.
0
0
0
0
0
0
25
0
0
5
10
0
3.3


G.
0
0
0
0
0
0
0
15
10
0
0
0
2.1


H.
0
0
0
5
10
0
30
35
40
10
10
15
12.9


I.
2
0
0
0
0
0
10
25
5
0
0
0
3.5


J.
25
25
10
5
15
25
30
0
40
40
40
20
22.9









Eleven days following the last treatment, disease severity was once again assessed. However, owing to the number of spots which made giving a numerical assessment impossible, disease severity was recorded using the following scale: 1-1—no disease, 2—slight, 3—moderate, 4—severe to 5—plant dead. The results are presented in Table 22.









TABLE 22







Treat-
Plant No.





















ment
1
2
3
4
5
6
7
8
9
10
11
12
Mean























A.
2
1
1
1
1
1
1
1
1
1
1
1
1.1 a


B.
2.5
2.5
1
4
3.5
3
4
2
2
3
3
3.5
2.8 c


C.
1
1
1
1
1
2
2
2.5
2.5
2
2
1
1.6 a


D.
1
1
2
1
2
1
1
1
1
1
2
2.5
1.4 a


E.
1
1
1
1
1
1
2
1
1
1
1
2
1.2 a


F.
1
2
2
1
1
1
2
1
1
1
2
1
1.3 a


G.
1
1
1
1
1
1
2
2
2
1
1
1
1.2 a


H.
2
2
1
2.5
2
2
2.5
3
3
2
2.5
2.5
2.2 b


I.
2.5
1
1
1
2
2
2
2
1
2
2
2
1.7 a


J.
3.5
4
3
3
3
4
3.5
2.5
4
4
4
4
3.5 d









As shown in Table 22, the bioactive compositions according to the present invention provided excellent protection against leaf spot, with those plants treated at the higher level and in combination with the commercial fungicide Eagle 40WP showing nearly the same level of disease as those that had not been inoculated at all. On the contrary, the Eagle alone, even at the recommended application rate, proved less efficacious than the bioactive composition. Finally, the Scala once again failed to show any efficacy and, in fact, proved more detrimental. It was suspected that the Scala treated plants manifested both leaf spot disease and phytotoxicity. None of the plants treated with the bioactive composition or the commercial Eagle fungicide showed evidence of phytotoxicity.
EXAMPLE 311
Fire Blight on Crabapples
A study of the bioefficacy of the various agrichemical compositions, including bioactive acid solutions and blends thereof with an antibiotic agrichemical, were evaluated to assess their bioefficacy against fire blight on crabapples. The study was conducted on 5 year old Snow Drift crabapple trees, with each of the compositions being applied to ten trees in two-subplots of five trees, four different times: at 100% bloom—day 1, a second application was made on day 4, a third on day 11 and a final treatment on day 19. The trees were inoculated with E. amylovora 153N at a concentration of 4×106 cells per milliliter on day 1, following drying of the treatment, with the inoculation being repeated on day 13. Evaluation for fire blight was completed on 100 blossoms for each of the subplots on day 12, day 19 and day 27. Additionally, the incidence of shoot infection and the length of cankers were evaluated on day 63. The results are shown in Table 23.
The test compositions were prepared by forming solutions of the bioactive agents. Two different dilutions of the MI2 bioactive were evaluated, the first employing 94.46 ml/gallon (25 ml/l) of water (5 ppm of each metal) and the second 188.9 ml/gallon (50 ml/l) of water (10 ppm of each metal). A streptomycin product (17% concentrated) was diluted at a rate of 0.049 lbs/gallon (58.72 g/l) (200 ppm). Finally, copper hydroxide was diluted at a rate of 1 lb/gallon (119.8 g/l). Each of the compositions and blends were spray applied at a rate of 50 gallons (189.3 l) pre acre. The specific tests and the results attained therewith are shown in Table 23. The results are presented as the mean of the counts.
As seen in Table 23, all tested compositions showed fewer infected flowers as compared to the control. The combination of the MI2 acid solution with streptomycin performed better than either bioactive alone. A significant rate response was seen between the two MI2 compositions: the 10 ppm solution performing significantly better than the 5 ppm solution. Shoot blight incidence was greatly reduced for the copper hydroxide, streptomycin an streptomycin/MI2 combination and a modest improvement with the higher concentrated solution of MI2. Only streptomycin and copper hydroxide appeared to prevent canker, while measurable cankers were noted with all compositions containing bioactive acid solution. Even so, these results show a marked benefit of the bioactive acid solutions alone or in combination with streptomycin. More importantly, it must be noted that even at the higher concentration MI2 composition, the 10 ppm of each metal, a total of 30 ppm metals, pales in comparison to the more than 50,000 ppm copper in the copper hydroxide solution. In essence, each application of the copper hydroxide release almost 1000 g or Copper into the environment compared to less than 2 grams or any one metal released by higher concentration MI2 composition.











TABLE 23






Rate
Blossom
Shoot
Canker


Bioactive
(per acre)
Incidence
Incidence
Size (cm)





















Day

12
19
27
63
63


MI2a
4.723 l/a
0.2
5.5
16.5
8.8
40.0


MI2
9.445 l/a
1.7
2.2
6.0
4.8
19.8


Copper
5 lbs/a
3.1
2.2
9.0
1.0
0.0


Hydroxideb
(2.27








kg/a)







Streptomycinc
0.49 lb/a
1.8
4.8
3.09
1.2
0.0


MI2 +
4.723/a
1.3
1.7
4.3
1.0
26.0


Streptomycin
0.49 lbs/a







Control
—
7.0
8.5
31.7
7.6
22.4


(untreated)





a16% w/w aqueous citric acid solution having dissolved therein silver citrate, copper citrate and zinc citrate, each added in an amount to provide 200 ppm of each metal, together with 0.25% sodium Lauroyl sarcosinate and 0.32% sodium lauryl sulfate.


b53% w/w concentrated copper hydroxide


c17% w/w streptomycin






Although the present invention has been described with respect to the foregoing specific embodiments and examples, it should be appreciated that other embodiments utilizing the concept of the present invention are possible without departing from the scope of the invention. The present invention is defined by the claimed elements and any and all modifications, variations, or equivalents that fall within the spirit and scope of the underlying principles.

## Claims

1. A method of killing, controlling and/or inhibiting the growth of fungi on plants said method comprising applying a bioactive composition to the soil in which the seed, crop or plant is or is to be planted; to the aqueous environment in which the plants are growing; or to the plants in their growing environment; said bioactive composition comprising, as a critical component thereof an efficacious antifungal active consisting essentially of a combination of i) at least one carboxylic acid selected from citric acid, valeric acid, itaconic acid, acetic, citriconic acid, lactic acid, malic acid, succinic acid, aldaric acid, malonic acid, proprionic acid, malonic acid, maleic acid, salicylic acid, glutaric acid, tartaric acids, and benzoic acid, ii) at least one antimicrobial metal ion or ion source, wherein the antimicrobial metal ions are selected from the group consisting of silver, copper, zinc, a combination of silver and zinc, a combination of silver and copper, a combination of copper and zinc and a combination of silver copper and zinc and iii) optionally, at least one surfactant, wherein the acid is present in the bioactive composition at a level of 0.01 to 10 weight percent and in at least a 2 times molar excess relative to the metal ions, the pH of the acid/antimicrobial metal ion combination in purified water is at least 2 and less than 6, and the antimicrobial metal ion is present in the bioactive composition in an amount of 500 ppm or less in the case of a single metal ion or 1000 ppm or less in the case or multiple metal ions, and wherein the rate of application of the bioactive composition to the soil or plants is such that no more than about 500 grams per acre of the metal ion(s) (determined as metal) is applied: said composition being free of and applied independent of any conventional antifungal bioactive agrichemical in an amount which manifests efficacious or synergistic antifungal activity or manifests synergy with the metal/acid antifungal active.

2. The method of claim 1 wherein the rate is such that the amount of bioactive acid composition derived antimicrobial metal ions being applied is from about 1 gram to about 250 grams per acre.

3. The method of claim 1 wherein the rate is such that the amount of bioactive acid composition derived antimicrobial metal ion being applied is from about 5 grams to about 100 grams acre.

4. The method of claim 1 wherein the bioactive composition is applied as an aqueous or aqueous-based solution, an oil in water emulsion, a wettable powder, a soluble powder, a suspension, a dust, granules or water dispersible granules, micro-capsules, gels, or tablets.

5. The method of claim 1 wherein the bioactive composition is applied directly to the plants and the pH of the acid/antimicrobial metal ion combination is such that little or no phytotoxicity occurs.

6. The method of claim 1 wherein the pH of the combination of the acid/antimicrobial metal ion in purified water is from 2 to 5.

7. The method of claim 1 wherein the antimicrobial metal ions are selected from a combination of silver and copper ions, a combination of silver and zinc ions, a combination of copper and zinc ions and a combination of silver, copper and zinc ions.

8. The method of claim 1 wherein the antimicrobial metal ions are a combination of silver, copper and zinc ions.

9. The method of claim 1 wherein the antimicrobial metal ion source is selected from organometallic compounds, antimicrobial metal salts, antimicrobial metal ion ion-exchange complexes and antimicrobial metal ion containing soluble glasses.

10. The method of claim 1 wherein the antimicrobial ion is present at a concentration of from about 1 ppm to about 300 ppm in the case of a single metal ion and from about 2 to about 500 in the case of multiple metal ions.

11. The method of claim 1 wherein the concentration of the antimicrobial ion in the bioactive acid composition is from about 2 ppm to about 100 ppm in the case of a single antimicrobial metal ion and from about 2 ppm to about 300 ppm in the case of multiple antimicrobial metal ions.

12. The method of claim 1 wherein the concentration of the antimicrobial ion in the bioactive acid composition is from about 5 ppm to about 50 ppm in the case of a single antimicrobial metal ion and from about 5 ppm to about 150 ppm in the case of multiple antimicrobial metal ions.

13. The method of claim 1 wherein the carboxylic acid is selected from citric acid, salicylic acid, glutaric acid and tartaric acid.

14. The method of claim 1 wherein the acid is present at a level of from about 0.1 to about 4 weight percent.

15. The method of claim 1 wherein the acid is present in a molar excess of at least 5 times.

16. The method of claim 1 wherein the at least one surfactant is present in an amount of from about 0.001 to about 3 weight percent.

17. The method of claim 16 wherein the at least one surfactant is an anionic, a non-ionic or an amphoteric surfactant.

18. The method of claim 1 wherein a combination of two or more surfactants is employed, each surfactant independently an anionic surfactant, a non-ionic surfactant or an amphoteric surfactant.

19. The method of claim 16 wherein a combination of two or more surfactants is employed, each surfactant independently selected from the group consisting of sulfonates, sulfates, sulfosuccinates, sarcosinates, mono-and di-glycerides, amine oxides, ether carboxylates, betaines, suflobetaines, and glycinates.

20. The method of claim 16 wherein the surfactants are independently selected from the group consisting of sultanates, sulfates, sulfosuccinates, sarcosinates, and amine oxides.

21. The method of claim 1 wherein the bioactive acid composition further comprises one or more conventional non-bioactive agrichemical additives selected from adjuvants, stabilizers, binders, oils for spraying, wetting agents, dispersing agents, emulsifiers, surfactants, fillers, rain fasteners, and dyes and other known active ingredients which have plant growth related properties.