GB2500381A

GB2500381A - The combination of pyrethroid and hydrogen peroxide for control of ectoparasite infestation in fish

Info

Publication number: GB2500381A
Application number: GB1204730.4A
Authority: GB
Inventors: Christopher Wallace; Raymond Lewis Waddell; Karl James Scott; David John Cockerill
Original assignee: MARINE HARVEST SCOTLAND Ltd
Current assignee: MARINE HARVEST SCOTLAND Ltd
Priority date: 2012-03-19
Filing date: 2012-03-19
Publication date: 2013-09-25
Also published as: GB201204730D0

Abstract

The combination of pyrethroid and hydrogen peroxide may be useful for control of ectoparasite infestation in fish. The pyrethroid may be deltamethrin, cypermethrin, cis-cypermethrin, cyfluthrin or tetramethrin. The ectoparasite infestation may be sea Louse (e.g. Lepeophtheirus salmonis or Caligus elongates). Apparatus for use in treatment of an aquaculture colony comprises a pyrethroid, hydrogen peroxide and at least one dosage device. The combination may be supra-additive in treating sea lice infestation in farmed fish (e.g. salmon). The hydrogen peroxide may contribute to the degradation of the pyrethroid (reducing environmental contamination).

Description

tM:;: INTELLECTUAL

PROPERTY OFFICE

Application No. 0B1204730.4 RTM Date:4 July 2012 The following terms are registered trademarks and should be read as such wherever they occur in this document:

ALPHA MAX

EXCIS

Intellectual Properly Office is an operaling name of Ihe Patent Office www.ipo.gov.uk

COMPOSITIONS FOR CONTROL OF ECTOPARASITES IN AQUACULTURE

FIELD

The invention relates to compositions for the control of ectoparasite infestation in aquaculture as well as methods of using such compositions.

BACKGROUND

With global capture fish stocks in decline the contribution of farmed fish to the food chain has seen consistent growth in recent decades. It is estimated that aquaculture contributes up to a third of the total global supply of aquatic food products (De Silva, Sena S. (2000) A global perspective of aquaculture in the new millennium, in Tech. Proc. of the Conference on Aquaculture in the Third Millennium, Network of Aquaculture Centres in Asia-Pacific, Bangkok, Thailand, pp. 431 -459). Given that seafood is the most highly traded food internationally, ensuring the security and sustainability of aquaculture is of vital importance to the global economy (Smith M et al. Science 12 February 2010: Vol. 327 no. 5967 pp. 784-786).

Fish farmed in sea pens may become infested by parasites from wild fish and may in turn become point sources for parasites. Sea lice are the best-studied example of this risk and are the most significant parasitic pathogen in salmon farming in Europe and the Americas. It is believed that parasitic infestations from sea lice cost the global aquaculture industry around 300 million a year in lost revenue. If left unchecked farmed fish will suffer and die. Hence, there is a need to address sea lice pressure with interventions which are useful to both remove and kill the parasite host leaving the host animal unaffected and safe for

introduction into the food chain.

Sea lice, copepods of the family Callgidae, are ectoparasites which thrive on the surface of wild marine fish. They are a primary cause of morbidity and impaired growth within farmed fish populations by feeding on the mucus, epidermal tissue, and blood of host animals. Sea lice are close relatives of other, better known, crustaceans such as shrimps and lobsters, and several hundred different species are found on fish throughout the world. In the UK, and in particular where farmed populations of fish such as Atlantic salmon are kept, two species of sea lice are commonly found: Lepeophtheirus salmon/s and Caligus elongatus. L. salmonis is generally found only on Atlantic salmon and sea trout, whereas C. elongatus is found on a much wider range of fish, including mackerel, herring as well as Atlantic salmon and sea trout. It is estimated that there are in excess of 150 different species of sea louse that will affect a wide variety of marine hosts.

Whilst there are a number of agents that are known to be effective for treatment of sea lice infestation, there is a growing appreciation of the environmental impact associated with use of such compounds. By way of example, in the UK there are severe regulatory controls governing discharge of so-called veterinary therapeutants into the environment. These regulations exist in order to protect the wider ecological systems in which aquaculture sites are often located, as well as to reduce the development of pesticide resistance in parasite populations. Use of organophosphate pesticides such as emamectin and dichlorvos has declined in recent years and become prohibited in many countries due to problems of environmental contamination and also increasing levels of resistance within sea lice populations.

In addition to organophosphates other known agents for treatment of sea lice on farmed fish include formaldehyde, hydrogen peroxide, chitin synthesis inhibitors (diflubenzuron and teflubenzuron), and pyrethroids (cypermethrin and deltamethrin).

There exists a need to provide improved therapeutant treatments for sea lice infestation in farmed fish that do not contribute inordinately to environmental pollution and yet are effective at overcoming parasite resistance. It is an object of the present invention to overcome the deficiencies in the art.

SUMMARY

The present inventors have identified a novel therapy for the treatment of sea lice in farmed fish populations that comprises a combination of a pyrethroid together with hydrogen peroxide. Surprisingly it has been found that not only do these two agents work together to reduce parasite infestation in a fish population, but the action of the hydrogen peroxide also contributes to degradation of the pyrethroid after effective use so as to significantly reduce the risk of wider environmental contamination.

A first aspect of the invention provides for pyrethroid and hydrogen peroxide for use in a combined treatment, or formulation, for control of ectoparasite infestation in a fish, wherein the treatment is characterised by the steps of exposing the fish to a composition comprising the pyrethroid and the hydrogen peroxide. Suitably, the pyrethroid is administered prior to, after or at approximately the same time as the hydrogen peroxide.

In a specific embodiment of the invention the pyrethroid comprises a synthetic pyrethroid or a pyrethrin. Suitably the synthetic pyrethroid is selected from one or more of the group consisting of: deltamethrin, cypermethrin; cis-cypermethrin; cyfluthrin and tetramethrin. In one embodiment of the invention the pyrethroid is administered substantially immediately prior to the hydrogen peroxide. In a further embodiment of the invention, the pyrethroid is administered at about the same time as the hydrogen peroxide.

In a specific embodiment of the invention, the combined treatment is administered in the form of a topical treatment. Suitably, the topical treatment comprises an immersion such as in a bath; vat or dip tank; well boat; or via a sluice or wash. Alternatively, the treatment may be administered directly to an aquaculture pen or a portion thereof.

In a further embodiment of the invention the pyrethroid is administered in an aqueous solution at a concentration of at least 0.01 milligrams per litre (mg/I) also referred to as parts-per-million, suitably at least 0.05 mg/I, more suitably at least 0.1 mg/I. At the upper end of the concentration an embodiment of the invention provides for pyrethroid at up to at most around 0.5 mg/I, suitably at most around 0.3 mg/I, and typically at most around 0.2 mg/I.

In a specific embodiment of the invention the hydrogen peroxide is administered in an aqueous solution at a concentration of at least 100 milligrams per litre (mg/I) also referred to as parts-per-million, suitably at least 250 mg/I, more suitably at least 750 mg/I. At the upper end of the concentration an embodiment of the invention provides for hydrogen peroxide at up to at most around 2000 mg/I, suitably at most around 1800 mg/I, and typically at most around 1500 mg/I.

In particular embodiments of the invention the time of exposure may vary from at least around 1 minute, typically at least around 5 minutes, more suitably at least around 10 minutes. At the upper end of the time exposure an embodiment of the invention provides for the maximum time to be around at most ninety minutes, suitably no more than around 45 minutes, typically around half an hour.

In accordance with a particular embodiment of the invention, the fish is comprised within an aquaculture colony -i.e. a fish colony. Suitably the fish may be selected from any farmed fish, including but not limited to salmon, trout, carp, sea bream, sea bass, cod, halibut, barramundi, cobia, grouper, red drum, turbot, catfish, bigeye tuna and tilapia. In a specific embodiment of the invention the fish is selected from salmon or trout -also referred to as a sa/monid. In further embodiments of the invention the combination therapy of the invention may be used for treatment of fish stocks held in recreational or scientific aquaria.

A second aspect of the invention provides a process for the removal of ectoparasitic infestation within an aquaculture colony comprising the steps of exposing the colony to a composition comprising a first agent and a second agent, wherein the first agent comprises a pyrethroid and the second agent comprises hydrogen peroxide. In a specific embodiment of the invention the first and second agents may be administered consecutively, concurrently or with an overlap of time.

A third aspect of the invention provides the use of a pyrethroid and hydrogen peroxide in the manufacture of a medicament for treatment of ectoparasite infestation in an aquaculture colony, wherein the treatment is characterised by the steps of exposing the colony to a composition comprising the pyrethroid and the hydrogen peroxide. In an embodiment of the invention the pyrethroid is administered prior to or at the same time as the hydrogen peroxide or with an overlap of time.

A fourth aspect of the invention provides apparatus, such as a kit, for use in treatment of an aquaculture colony, the apparatus comprising a first therapeutic agent, a second therapeutic agent and at least one dosage device, wherein the first therapeutic agent comprises a pyrethroid and the second therapeutic agent comprises hydrogen peroxide. In a specific embodiment the first and second therapeutic agents are provided in concentrated form. In typical embodiment of the invention the dosage device comprises a measuring cylinder, a container with measuring scale, a dropper, a syringe, or a container of known volume (such as a cup or vessel). Suitably the first and second therapeutic agents may be comprised in separate containers either of which further comprises an integrated dosage device.

DRAWINGS

The invention is illustrated with reference to the following drawings in which: Figure 1 shows a graph illustrating that lice removed from fish after a hydrogen peroxide treatment and placed in ambient sea water conditions can survive and remain active for 45 days off host.

Figure 2 shows a graph demonstrating the survival of gravid/adult female sea lice in days following Alphamax'TM (deltamethrin) treatment in ambient sea water.

Figure 3 shows a graph of results of a first experimental field trial of the invention.

Figure 4 shows a graph of results of a second experimental field trial of the invention.

Figure 5 shows a graph of results of a third experimental field trial of the invention.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Sea lice of the Cal/gidae species, including Lepeophtheirus salmon/s and Galigus elongates, have a complex life cycle that includes periods when the organism is free swimming (planktonic) as well as other times where it is attached to a host (parasitic). Within these stages are many sub-stages that are separated by moults as the organism grows and develops from a larval stage through to full adulthood. The development rate for L. salmon/s from egg to adult varies from between 17 to 72 days depending on temperature and host availability. The relatively controlled environment associated with farmed fish, especially the high density of host animals, present ideal conditions for sea lice to thrive.

In accordance with the present invention, a combined treatment is provided by way of a controlled dosage regimen that comprises exposure of fish that require treatment to a pyrethroid and hydrogen peroxide. The combined and supra-additive properties of these agents serve to reduce levels of ectoparasite infestation in fish aquaculture colonies as well as to prevent re-infection over the longer term. Hence, they are suitably used in a combination therapeutant for treatment of ectoparasite infestation.

As used herein the term therapeutant refers to chemical substances used in aquaculture operations or in aquaria when necessary to keep aquatic animals, suitably fish, healthy while they are being raised.

Pyrethrum is the mixture of cyclic compounds found in chrysanthemum flowers and includes six chemicals with insecticidal properties (pyrethrins). Pyrethroids are synthetic esters derived from pyrethrins and have well characterised toxicity to arthropods including insects and crustaceans. Synthetic pyrethroids share a common chemical structure consisting of cyclopropane carboxylic acids, with variations in the alcohol portion of the compounds. Pyrethroids are divided into two different groups: Type I pyrethroids, which do not contain a cyano group and Type II pyrethroids which contain a cyano group added to the benzylic carbon atom. Pyrethroids act as sodium channel blockers in arthropods and are highly neurotoxic to invertebrates. In spite of their known toxicity, several pyrethroid resistant populations of sea lice have been identified with genetic mutations of the sodium channels that render the compounds less effective, particularly at working therapeutant concentrations.

Exemplary pyrethroids with known toxicity to fish ectoparasites, such as sea lice, include deltamethrin, cypermethrin; cis-cypermethrin; cyfluthrin and tetramethrin. However, it will be appreciated that these and other synthetic pyrethroids and derivatives with equivalent properties may be used in the present invention either alone or in combination. In an exemplary embodiment of the present invention, described in more detail below, the pyrethroid deltamethrin is used in combination with hydrogen peroxide in order to provide topical treatment for sea lice infestation in salmonids.

Deltamethrin is commonly sold under the brand name AlphaMaxTM (Pharmaq AS, Overhalla, Norway), whereas cypermethrin is sold under the brand name ExcislM and cis-cypermethrin as BetamaxlM (Vericore Ltd., UK). The chemical structure of deltamethrin is shown in Formula I below:

I

The chemical structure of cypermethrin is shown in formula II below: Gl' ° H

N

Hydrogen peroxide has been known to be effective in treatment of sea lice particularly during mobile and attached (chalimus) stages of development. It is believed that the main mode of action is by the generation of lethal oxygen emboli within the bodies of the sea louse. In addition, hydrogen peroxide appears to stimulate disengagement of attached sea lice from the host as well as sterilization of egg strings. It should be noted, however, that hydrogen peroxide has a narrow safety window and is a powerful oxidising agent which can cause toxicity to fish if used in excess and at higher water temperatures.

It is surprising, therefore, that the combined action of two known pesticide agents can be supra-additive in treating sea lice infestation in farmed fish. It is also surprising, given that resistance to at least one of the agents -pyrethroid -is known to be prevalent within sea lice populations. Without wishing to be bound by theory it is believed by the inventors that combined and synergistic activities of these two agents serve to stimulate the disengagement of attached sea lice from the surface of the fish which then exposes the lice to the toxic actions of both agents in combination. Since it is shown in the accompanying examples that sea lice are capable of living for up to 45 days away from a host (see Figures 1 and 2), it is important that any disengaged parasites are killed within a short period after disengaging, otherwise re-infection of the treated fish is likely to occur.

A further advantage of the present invention is that the oxidising activity of hydrogen peroxide also hastens degradation of the pyrethroid, thereby reducing wider environmental contamination as well as the opportunity for development of further resistance within nearby sea lice populations that have not been directly treated.

The treatment of the invention is typically administered to one or more fish by way of short-term exposure in a controlled enclosure. The enclosure may be in the form of a chamber, tank, vat or well that is isolated from the main containment area (i.e. fish pen). In an alternative embodiment of the invention the treatment is administered to a semi-segregated zone within the main containment area, such as by the use of sheeting, booms or baffles. The time of exposure will vary depending upon the level of infestation, water temperature and other environmental considerations. However, it is typical that the treatment phase lasts at least one minute and at most 90 minutes. As mentioned, both components of treatment may be administered simultaneously or sequentially.

Suitably, where sequential administration occurs the pyrethroid can be administered before or after the hydrogen peroxide. If it is intended to enhance degradation of the pyrethroid following treatment then it is suitably administered before the hydrogen peroxide. In certain embodiments of the invention sequential administration of the first and second components will overlap such that at least a portion of the treatment will involve simultaneous exposure.

The invention provides a process for the removal of ectoparasitic infestation within an aquaculture colony comprising the steps of exposing the colony to a composition comprising a first agent and a second agent, wherein the first agent comprises a pyrethroid and the second agent comprises hydrogen peroxide. In a specific embodiment of the invention the first and second agents may be administered consecutively, concurrently or with an overlap of time. It will be understood that this embodiment refers to either the eradication of infestation from the bodies of fish comprised within the aquaculture colony, or alternatively to apparatus, facilities and aspects of plant that are found within a typical aquaculture colony where detached or free-swimming sea lice may reside. For example, in certain instances it is necessary to run sterilization procedures for otherwise empty tanks or pens prior to introduction of any new fish. In such instances the treatments, apparatus and compositions of the invention may be used in a prophylactic manner to prevent infestation from occurring.

A specific embodiment of the invention provides apparatus, such as in the form of a kit, for use in treatment of an aquaculture colony. The kit can comprise first therapeutic agent and a second therapeutic agents and at least one dosage device, wherein the first therapeutic agent comprises a pyrethroid and the second therapeutic agent comprises hydrogen peroxide. The first and second therapeutic agents are suitably provided in concentrated form, that is in a form that is appropriate for transport and dilution to a working concentration at the site where therapy is to occur. Suitably the first and second therapeutic agents may be comprised in separate containers either of which further comprises an integrated dosage device. In typical embodiment of the invention the dosage device comprises a measuring container, such as a graduated cylinder or such like. Alternative containers with measuring scales may be used. The containers may comprise integrated or separate droppers, syringes, or containers of known volume (such as a cup or vessel). The size and arrangement of the dosing device will vary dependent upon the intended market for the apparatus. For example, a kit for use by a domestic aquarium hobbyist will be significantly smaller in scale than that suitable for use with an in-shore salmon fishery. It will be appreciated by the skilled person that the various components of such apparatus can be scaled up and down as appropriate to achieve the necessary application. Apparatus of the invention may also comprise additional components including protective equipment to assist handling, instructions for use and optionally assay components to assist with monitoring of pH, temperature or dissolved oxygen levels.

The invention is further exemplified with reference to the following non-limiting

example.

EXAMPLES

Here we document a combination therapeutant against sea lice, using the deltamethrin-based AlphamaxTM composition (Pharmaq AS) and a preparation of 35% hydrogen peroxide (Brenntag NV, Deerlijk, Netherlands) in 3 different trials. The use and effects of hydrogen peroxide are well documented but the present research suggests that whilst sea lice egg strings are generally impaired by the product at lSOOppm (1500 mg/I), there is a likelihood of reinfestation from removed lice which have not been permanently impaired by the action of H202. AlpharnaxTM is also well tried and tested within the industry with varied and occasional concerns over the dispersion of the medicine within the treatment enclosure. It is also noted that AlphamaxlM has a delayed action in louse removal, with dead lice remaining attached to fish. This often makes assessment of treatment efficacy suspect in the eyes of the non-professional. In combining these two modes of action we have identified surprisingly a treatment which kills lice quickly whilst also yielding clean parasite-free fish.

Example 1

Figure 1 shows the results of an experiment in which salmon known to be infected with Lepeophtheirus.salmonis were treated in a bin with hydrogen peroxide at a concentration of 1750 mg/L (ppm) in ambient sea water. Detached sea lice were kept in ambient sea water (11°C) and inspected periodically to determine viability. It is evident that adult female lice were able to survive in significant numbers up to 45 days away from a host.

Example 2

The results of an experiment are shown in Figure 2, in which salmon known to be infected with L. salmon/s were treated in a 500 litre capacity bin in sea water with 0.2 ml deltamethrin (Alphamax'TM). Detached sea lice were kept in ambient sea water (11°C) and inspected periodically to determine viability. It is evident that adult female lice were able to survive in significant numbers up to 10 days after treatment and that for the course of the experiment complete eradication was not achieved.

The results of Experiments 1 and 2 indicate that individually hydrogen peroxide and pyrethroid are effective in reducing sea lice infestation in an initial phase but do not eradicate parasites over the mid-to longer-term.

Example 3

Outlined below are the results of three trials using combinations of pyrethroid (AlphamaxTM) and hydrogen peroxide as a topical treatment against L. salmon/s on 2.5 kg atlantic salmon at MHS Ardnish Salmon Farm, Lochailort, Inverness-shire.

In each treatment seven new fish were placed into a treatment tank containing 500 litres of fresh sea water at 11°C. Fish were subjected to the combined treatment and then anaesthetised using 100mg/I (ppm) MS222 (Fharmaq AS) for lice removal. After 30 minutes of topical treatment fish were anaesthetised, placed in a separate bin and any lice present were removed to a container containing fresh seawater for observation.

Water remaining in the bins was passed through a 0.5 mm2 mesh screen. In trials 2 and 3 no lice were found in the bottom three quarters of the water column of either bin. Lice either filtered from the top quarter or picked off the sides were removed to a container of fresh seawater for observation. Lice were assessed for viability at 24 (1+24) and 48 (1+48) hours post-treatment.

Figure 3 shows the results of the first trial using AlphamaxTM (0.2m1 per 500 litres) for 10 minutes followed by 20 minutes of 1000 mg/I (ppm) hydrogen peroxide giving a total topical treatment time of 30 minutes.

Figure 4 shows the results of the second trial using hydrogen peroxide at 500 mg/I (ppm) for 10 minutes followed by AlphamaxTM (0.2m1 per 500 litres) for 20 minutes giving a total topical treatment time of 30 minutes.

Figure 5 shows the results of the third trial using hydrogen peroxide at 500 mg/I for 20 minutes followed by AlphamaxTM (0.2m1 per 500 litres) for 10 minutes giving a total topical treatment of 30 minutes.

Hence, the trials demonstrate the effectiveness of combining pyrethroid with hydrogen peroxide treatments in therapeutants for rapid eradication of sea lice infestation in farmed fish. The combined actions of these two agents are considerably more effective than when the substances are used alone. In addition, the added benefit that the combined treatment undergoes more rapid degradation leads to reduced environmental impact.

Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the appended claims, which follow. The choice of fish stock or the actual therapeutant agents of interest are believed to be a routine matter for the person of skill in the art with knowledge of the presently described embodiments. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

Claims

CLAIMS: 1. Pyrethroid and hydrogen peroxide for use in a combined treatment for control of ectoparasite infestation in a fish, wherein the treatment is characterised by the steps of exposing the fish to a composition comprising the pyrethroid and the hydrogen peroxide.
2. The treatment of claim 1, wherein: (a) the pyrethroid is administered prior to the hydrogen peroxide; or (b) the pyrethroid is administered at the same time as the hydrogen peroxide; or (c) the pyrethroid is administered after the hydrogen peroxide.
3. The treatment of claims 1 and 2, wherein the pyrethroid comprises a synthetic pyrethroid or a pyrethrin.
4. The treatment of claim 3, wherein the synthetic pyrethroid is selected from one or more of the group consisting of: deltamethrin, cypermethrin; cis-cypermethrin; cyfluthrin and tetramethrin.
5. The treatment of any previous claim wherein the treatment is administered topically.
6. The treatment of any previous claim wherein the treatment is administered via immersion, such as in a bath; vat or tank; well boat; or via a sluice or wash.
7. The treatment of any previous claim wherein the treatment is administered directly to an aquaculture pen or a portion thereof.
8. The treatment of any previous claim wherein the pyrethroid is administered in an aqueous solution at a concentration of at least 0.01 milligrams per litre (mg/I), optionally at least 0.05 mg/I, more suitably at least 0.1 mg/I.
9. The treatment of any previous claim wherein the pyrethroid is administered in an aqueous solution at a concentration of at most around 0.5 mg/I, optionally at most around 0.3 mg/I, and typically at most around 0.2 mg/I.
10. The treatment of any previous claim wherein the hydrogen peroxide is administered in an aqueous solution at a concentration of at least 100 milligrams per litre (mg/I), optionally at least 250 mg/I, suitably at least 750 mg/I.
11. The treatment of any previous claim wherein the hydrogen peroxide is administered in an aqueous solution at a concentration of up to at most around 2000 mg/I, optionally at most around 1750 mg/I, and typically at most around 1500 mg/I.
12. The treatment of any previous claim wherein the time of exposure is from at least around 1 minute, optionally at least around 5 minutes, suitably at least around 10 minutes.
13. The treatment of any previous claim wherein the time of exposure is for a maximum time of around at most ninety minutes, optionally no more than around 45 minutes, typically around half an hour.
14. The treatment of any previous claim wherein the fish is comprised within an aquaculture colony.
15. The treatment of any previous claim wherein the fish is selected from any farmed fish, including but not limited to salmon, trout, carp, sea bream, sea bass, cod, halibut, barramundi, cobia, grouper, red drum, turbot, catfish, bigeye tuna and tilapia.
16. The treatment of claim 15, wherein the fish is selected from salmon or trout.
17. The treatment of any previous claim wherein the ectoparasite is a sea louse, optionally selected from Lepeophtheirus sa/monis or Cal/gus elongates.
18. A process for the removal of ectoparasitic infestation within an aquaculture colony comprising the steps of exposing the colony to a composition comprising a first agent and a second agent, wherein the first agent comprises a pyrethroid and the second agent comprises hydrogen peroxide.
19. The process of claim 18, wherein the first and second agents are administered consecutively, concurrently or with an overlap of time.
20. Apparatus for use in treatment of an aquaculture colony, the apparatus comprising a first therapeutic agent, a second therapeutic agent and at least one dosage device, wherein the first therapeutic agent comprises a pyrethroid and the second therapeutic device comprises hydrogen peroxide.
21. The apparatus of claim 20, wherein the first and second therapeutic agents are provided in concentrated form.
22. The apparatus of claims 20 and 21, wherein the dosage device comprises a measuring cylinder, a container with measuring scale, a dropper, a syringe, or a container of predetermined volume.
23. The apparatus of claims 20 to 22 wherein the first and second therapeutic agents are comprised within separate first and second containers.
24. The apparatus of claim 23, wherein either or both of the first and second containers further comprises an integrated dosage device.