CA2290822A1 - A microbiocidal formulation - Google Patents

Abstract

The invention concerns a microbiocidal formulation suitable, following dissolution in a diluent, to form microbiocidal solution for microbiocidal treatment of an environment. The formulation comprising sufficient diluent alkalinity neutralising agent so that, following dissolution in the diluent, an alkalinity of no more than 100, preferably no more than 50 mg/l bicarbonate alkalinity is observed in the microbiocidal solution. The formulation also comprises sufficient pH neutralising agent so that, following dissolution in the diluent, a pH of 5.0-8.0, preferably 6.0-6.8, is observed in the microbiocidal solution. The formulation also comprises a microbiocidally effective amount of an alkalinity sensitive microbiocidal agent. The formulation is adapted to release the microbiocidally effective amount of the alkalinity sensitive microbiocidal agent over a microbiocidally effective period of time.

Description

A MICROBIOCIDAL FORMULATION
The present invention relates to a microbiocidal formulation for dissolution in water as a diluent to form a microbiocidal solution, said formulation containing an alkalinity neutralising agent to neutralise the total alkalinity present in the water used as the diluent.
It is well known how to produce a whole range of biocides, microbiocides and disinfectants from halogen donors, particularly those of chlorine and iodine.
Typical of such donors are sodium and calcium hypochlorite, chloramines, iodophors and sodium dichloroisocyanurate (NaDCC). While the effectiveness of all of these donors as a source of microbiocidally effective agent, e.g. hypochlorous acid, has been shown in various laboratory trials at different concentrations of the agent against a wide range of micro-organisms, it has been found that, in the field, the microbiocidal efficiency was not as expected from the laboratory trials.
After detailed investigation and further tests, it was surprisingly learnt that the factor most influencing the effectiveness of the microbiocidal solution was the water used in its preparation.
The invention provides a microbiocidal formulation suitable, following dissolution in a diluent to form a microbiocidal solution for microbiocidal treatment of an environment, the formulation comprising sufficient diluent alkalinity neutralising agent so that, following dissolution in the diluent, an alkalinity of no more than 100, preferably no more than 50 mg/1 bicarbonate alkalinity is observed in the microbiocidal solution; sufficient pH neutralising agent so that, following dissolution in the diluent, a pH of 5.0-8.0, preferably 6.0-6.8; is observed in the microbiocidal solution and a microbiocidally effective amount of an alkalinity sensitive microbiocidal agent; the formulation being adapted to release the microbiocidally effective amount of the alkalinity sensitive microbiocidal agent over a microbiocidally effective period of time.
Preferably, the alkalinity sensitive microbiocidal agent is a source of available halogen.
More preferably, the formulation is adapted to release available halogen, in use, from an organic source/precursor of hypohalous acid and/or hypohalite.
Advantageously, the organic source/precursor compound releases, in aqueous solution, hypohalous acid and/or hypohalite in a microbiocidally effective amount over the microbiocidally effective period of time, by adaption of the formulation for the control of the pH.
More advantageously, the alkalinity neutralising agent is comestibly acceptable.
Preferably, the organic source/precursor compound is a halogenated isocyanuric compound or a salt thereof.

More preferably, the source/precursor compound of the microbiocidally effective hypohalous acid and/or hypohalite is selected from sodium dihaloisocyanurate, potassium dihaloisocyanurate or trihaloisocyanuric acid, preferably sodium dichloroisocyanurate.
Even more preferably, the microbiocidally effective amount of the hypohalous acid is released from 1 to 5000 ppm of the solid source/precursor compound and the microbiologically effective period of time is in the range of 10 seconds to 48 hours.
Advantageously, the micro-organism is selected from E.coli or Pseudomonas or is a resistant micro-organism, more preferably a pasteurisation resistant micro-organism, still more preferably a thermoduric or thermophilic organism, most preferably, a species of micro-organism selected from Bacillus, Micrococcus, Microbacterium, Clostridium, Listeria, AZcaliigenes, Arthrobacter, Lactobacillus, Serratia or any other spore forming species.
More advantageously, the environment comprises an external surface or a lumen of an apparatus used in the production, preparation or processing of food or beverages.
Even more advantageously, the environment comprises process - liquid or liquid for human or animal consumption.
Preferably, the alkalinity neutralising agent is a comestibly acceptable acid or its salt or a mixture thereof, preferably a comestibly acceptable organic acid.

More preferably, the alkalinity neutralising agent is succinic acid or a salt thereof.
Even more preferably, the alkalinity neutralising agent is citric acid or a salt thereof.
Advantageously, the microbiocidal formulation is in powder, granulate or tablet form.
It is postulated that alkalinity affects the microbiocidal activity of the hypohalous acid and/or its hypohalite salts by neutralising the hypohalous acids themselves and by affecting the pH of the microbiocidal formulation in the environment.
Upon investigation of the normal or "field" water used as the diluent, as opposed to the distilled water commonly used in laboratory trials, bicarbonate alkalinity or total alkalinity was identified as the cause of the reduced effectiveness of the microbiocidal solution. This explains why the field results were disappointing when compared to the laboratory tests.
It was found that dissolved bicarbonate mineral salts were combining with the dissociation product of the halogen donor and reducing its availability for microbiocidal action. This was particularly noticeable where the microbiocidal solution had to act at a low (low freely available chlorine concentration) level in a large volume of diluent water. The larger the volume/proportion of diluent water compared to the microbiocide, the more dissolved bicarbonate salts were available to combine with the microbiocide agent and to significantly reduce its effectiveness.
Most surprisingly, this factor does not appear to have been 5 taken into account in the prior art and the applicants are unable to find prior art on the relative microbiocidal effectiveness of known microbiocides in field conditions of varying diluent total alkalinity.
Tests were conducted to compare laboratory trials using distilled water or water of a known alkalinity as the diluent. These comparisons provided new and very surprising results with regard to the efficiency of the microbiocidal agent when dissolved in high alkalinity water as the diluent.
In order to understand how the total alkalinity of "field"
water could impact on microbiocidal activity, a study was undertaken to understand what "total alkalinity" or ANC (acid neutralising capacity) actually was.
Alkalinity is the sum of all titratable bases and is a measure of the acid neutralising capacity (ANC). Water total alkalinity is mainly a sum of carbonate, bicarbonate and hydroxide levels but may also include contributions from borates, phosphates, silicates or other bases, if these are present. Bicarbonates are the main contributors to "field"
water total alkalinity.
Alkalinity of water is pH related but is not pH dependant, for example, water can have a pH of 6.4 and an alkalinity of ~ ~
w~ ~~ ~~~ ~~ ~~ ~~ ~~ ~~
~ ~ ~ ~ ~~ ~ ~ ~w~ ~ ~ ~ ~
~ 1 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ w w ~ ~ ~ ~ ~ 1 ~ 1 ~ ~ 1 ~ ~ ~ ~ ~ ~ ~ 1 ~ ~ ~ ~ ~ 1 320 mg/1 and, conversely, water can have a pH of 8.0 and an alkalinity of 95 mg/1. Reference is made to Figure 4 of the accompanying drawings showing the lack of relationship between pH and total alkalinity of various field waters obtained in Ireland.
Geological factors are a major influence on the levels of total alkalinity likely to be found in water supplies. Thus, the greater the water residence time in a limestone/dolomite region, the greater the likelihood of a high alkalinity.
Conversely, water from granite/sandstone areas will have a low or lower total alkalinity.
Some of the definitions used herein have quite specific meanings in relation hereto and are now explained.
The expression "ANC" means "acid neutralising capacity".
This term is now coming into use to replace the classical expression "total alkalinity", although the latter remains the most widely used term in the field of applied water chemistry. Outside the field of applied water chemistry, total alkalinity as a concept separate from pH is not at all well understood.
The expressions "FAH" and "FAC" mean "freely available halogen" and "freely available chlorine", respectively, that is, halogen or chlorine in a microbiocidally active form.
Concerning chlorine, the total concentrations of chlorine, hypochlorous acid and hypochlorite ion are the "FAC" of the microbiocidal solution - except at extremely low pH
conditions, the chlorine can be ignored. Figure 1 of the c~~~ AMENDED SHEET
wJ..
~iZ~.

accompanying drawings illustrates the pH distribution curves for each of the three FAC species (it is necessary to specify a CI concentration of l.OmM and a temperature of 28°C.
Figure 2 shows a simplified pH distribution curves for the FAC species, based on reasonably ignoring the significant C1z contribution when the pH is greater than 3 (it is not now necessary to specify the C12 concentration). Figure 3 shows the pH dependence of these two FAC species, in which the respective percentages are on a molar basis.
By example, a chlorine donor compound is often referred to as having say 500mg of available chlorine per gram. This describes the total amount of chlorine available but describes neither the form, its activity, nor its availability at a given time, all of which will affect the microbiocidal effectiveness of the solution.
Lubricating agents are commonly used in the tabletting process e.g. adipic acid or succinic acid. However, their sole disclosed function to date has been as a lubricating agent or as the replacement of an existing lubricating agent.
No attempt had been made in the prior art to adapt these compounds or the like to act as a neutralising agent with regard to the impact of bicarbonate alkalinity on the efficacy of the microbiocidal agent in any given microbiocidal solution.
Many other compounds other than those identified above can be introduced to perform this function such as, for example, any comestibly acceptable acid or a salt thereof. However, where practical, the level of a suitable lubricating agent should be adjusted to accommodate or neutralise the impact of the total alkalinity or ANC value of the diluent water on the microbiocidal effectiveness of any given microbiocidal solution.
Cognisant of this, the lubricating component has been taken into account when calculating the adjustment that would be required to a "conventional" microbiocidal formulation to neutralise the total alkalinity or ANC of any given diluent water. As previously stated, total alkalinity is pH related but not pH dependant and the use of a comestible acid to neutralise the alkalinity had the additional benefit of achieving an optimum pH for the dissociation of the chlorine donor NaDCC. Thus, a comestible acid (or a salt thereof) was selected for further tests.
As the object of the present invention was to neutralise (reduce or eliminate) the ANC of the diluent water, once the formulation was added to the diluent water and full dissolution had taken place, the dosed water (formulation dissolved in diluent) achieved the target dosed pH of 5.0-8.0, preferably, 6.0-6.8 and the target dosed ANC of no more than 100 mg/1 bicarbonate alkalinity, preferably no more than 50 mg/1 bicarbonate alkalinity.
An NaDCC based tablet was prepared containing, along with various components customarily employed to facilitate tabletting and to promote dissolution of the tablet by means of effervescence, a comestible agent present in a specifically adjusted amount to achieve a desired target pH

and FAC (free available chlorine) value in the dosed water when the tablet was completely dissolved in the diluent water. Dissolution requires that all the tablet components are uniformly distributed in the diluent, that the solution is fully diluted to the required dilution for its use as a microbiocidal agent and that following partial or virtually complete loss of the excess dissolved carbon dioxide introduced by the effervescence.
The comestible acid (or a salt thereof) may be a comestible agent additional to "conventional" tabletting agents and/or effervescence agents, but the amount of comestible agent must be specifically determined to attain the desired pH and FAC
value, following loss of excess dissolved carbon dioxide to a contiguous gaseous phase.
The amount of comestible agent required to produce the desired pH and FAC value in the dosed water is determined by a calculation which takes account of the following factors:
(i) The pH of the diluent water;
(ii) The acid neutralising capacity (ANC), classically referred to as the total alkalinity (as defined by APHA Standard Methods), of the diluent water;
(iii) The amount of halogen donor such as NaDCC, together with the amounts of the other dosants of a formulation, to be added to a unit volume of the diluent water;

(iv) The numerical values of the complete set of equilibrium constants appropriate to the conditions of the diluent water and defining the equilibrium conditions of the set of interconverting chemical 5 species derived from the halogen donor, such as NaDCC, the chemical species derived from the carbonate and bicarbonate salts, the chemical species derived from any lubricating agents (including comestible acids incorporated for that 10 purpose) in the formulation, and the chemical species derived from the comestible acid which is to serve as the pH- and FAC-adjusting species as defined hereinabove;
(v) The pH desired for the dosed water following complete dissolution of the formulation, following partial or virtually-complete loss of the excess carbon dioxide, which is defined as a "final or ultimate degree of saturation with respect to carbon dioxide" in paragraph (vii) below;
(vi) The free available halogen donor or chlorine (FAH
or FAC) concentration or, alternatively, the hypochlorous acid concentration or, alternatively, the hypochlorite ion concentration, desired for the dosed water following complete dissolution of the formulation and loss of excess dissolved carbon dioxide as described in paragraph (v). The desired final FAC or hypochlorous acid or hypochlorite ion concentration may be prescribed [but are not essentially prescribed] as a fraction of the total concentration of chlorine (C1T) introduced into the diluent water, at its desired dilution, by the NaDCC component of the formulation (C1T, expressed as mol per unit volume, is twice the number of mols of NaDCC added to the unit volume of the diluent).
(vii) The "degree of saturation with respect to carbon dioxide", referred to in paragraph (v), is the concentration of free carbon dioxide in a water, in this case, the dosed and fully diluted water, expressed as a fraction of the concentration of free carbon dioxide which would be present if the water was at equilibrium with a contiguous gaseous phase for which the partial pressure of carbon dioxide is known or prescribed;
(viii) The concentration of "free" carbon dioxide, referred to in paragraph (vii), can be interpreted as the concentration of C02 per se [as opposed to the concentration of "dissolved" carbon dioxide which embraces the dissolved molecular species CO2, in addition to carbonic acid (HzC03) and its dissociation products, the bicarbonate (HC03) and carbonate ions ( C032 ) ] .
(ix) The numerical value of the "degree of saturation with respect to carbon dioxide", referred to in paragraph (vii), is obtained by multiplying the partial pressure of carbon dioxide in the gaseous phase by the value of the Henry's law constant for carbon dioxide for the condition of the water and for "free" carbon dioxide in accordance with the alternatives described in paragraph (viii).
Investigation of the impact of total alkalinity or ANC on microbiocidal efficacy in an effervescent tablet formulation containing a commonly used active ingredient as a halogen donor (NaDCC) clearly demonstrates that, when alkalinity is taken into consideration and an agent to neutralise same is added to the formulation, there is an immediate improvement in the microbiocidal efficiency as shown in comparative trials described in the following examples. These laboratory trials were carried out with three chlorine donors, as the chosen halogen, because chlorine is a commonly used halogen in commercial microbiocides, namely, sodium hypochlorite; a commercial NaDCC based product, AgriseptR Tabs; and a test product, the same formulation as AgriseptR Tabs, but containing, in addition, an agent to neutralise the diluent alkalinity.
Typically, if the above-mentioned calculation is applied, then, as two critical factors are varied, so too does the required quantity of the alkalinity neutralising agent vary.
It has been found that the two factors most likely to be altered by field conditions are total alkalinity or ANC of the diluent water; and the volume of the diluent water;
It is clear that, when the ANC increases, then the required amount of the alkalinity neutralising agent also increases.
However, it should be remembered that, when the volume of diluent water is increased, the total amount of ANC in the water also increases, whereas the amount of alkalinity neutralising agent in any given tablet formulation is fixed.
By use of a predictive computer programme based on the above-mentioned factors (i) to (ix), it is possible to calculate how to vary the quantity of two potential alkalinity neutralising agents, citric acid and succinic acid, so as to achieve the target dosed ANC value using a specified volume of a diluent and using different volumes of a diluent.
Table 1 shows, using this predictive computer programme, the impact of altering the diluent's ANC, whilst maintaining all other factors constant. The formulation comprises (excluding the alkalinity reducing agent) 2.218 NaDCC, 1.138 adipic acid, 1.0368 sodium bicarbonate and 0.0448 sodium carbonate.
The diluent water pH is 7.5 and its volume is 11. The target dosed pH and FAC to total C1 ratio are 6.0 and 0.5, respectively.
Table 1 Diluent Calculated Alkalinity Amount of Alkalinity Total Carbonic Neutralising Succinic or (mg/1 as Carbon Agent Citric Acid calcium (mmol/1) (/tablet) Required carbonate) (g/tablet) 100 2.1 0.9 0.034 200 4.2 3.7 0.171 400 8.5 9.2 0.445 Table 2 shows, using the predictive computer programme, the impact of altering the diluent volume, whilst maintaining all other factors constant. The formulation comprises (excluding the alkalinity neutralising agent} 2.218 NaDCC, 1.138 adipic acid, 1.0368 sodium bicarbonate and 0.0448 sodium carbonate.
The diluent water pH is 7.5 and its volume is 101. The target dosed pH and FAC to total Cl ratio are 6.0 and 0.5, respectively.
Table 2 Diluent Calculated Alkalinity Amount of Alkalinity Total Carbonic Neutralising Succinic or (mg/1 as Carbon Agent Citric Acid calcium (mmol/1) (/tablet) Required carbonate) (g/tablet) 25 0.5 5.1 0.239 100 2.1 22.3 1.267 200 4.2 37.4 2.638 400 8.5 54.9 5.379 Table 3 shows the impact of altering the amount of adipic acid (the conventional lubricating agent), whilst maintaining all other factors constant. The composition of the formulation is as set out above for Tables 1 and 2. The diluent water volume is 11, its pH is 7.5 and its total alkalinity is 400 mg/1. The target dosed pH and FAC to total C1 ratio are 6.0 and 0.5, respectively.

Table 3 Adipic Acid Amount of Succinic or Total Succinic Acid Citric Acid Alkalinity or Citric (/tablet) Reducing Acid Required Agent (g/tablet) (/tablet) g/tablet o/tablet 1.0 20.6 0.561 11.6 32.2 1.25 25.6 0.339 6.9 32.5 1.50 30.6 0.117 2.4 33.0 The impact on microbiocidal efficiency of halogen-based 5 microbiocides, in particular, and, more generally, of any microbiocide whose activity in solution is influenced or degraded by the ANC value of the diluent water is as follows:
Table 4 [Microbiocide] Diiuent Volume [ANC] Impact Example High Medium Low Little 7, 9 Low Low Low Medium High High Low Medium Low High Low Major 11 High Medium High Medium 8 Low Low High Major 12 High High High Medium 13 Low High High Major 10 It will be appreciated that the microbiocidal formulation of the present invention is particularly applicable to neutralising the medium and major negative impacts set out hereinabove and is most particularly applicable to neutralising the major negative impacts set out hereinabove.
The invention will now be understood in greater detail from the following description of preferred embodiments thereof given by way of example only.
A microbiocidal formulation of the present invention has the following preferred composition:
Ingredient Unit o By Function Reference Weight Per to Tablet Standards Succinic Acid 53.1 Reduces alkalinity EP

Sodium Dichloro- 23.5 Source releasing HSE

isocyanurate hypohalous acid tanhydrous) into solution Sodium 11 Carbon dioxide EP

bicarbonate yielding component of effervescent base Adipic Acid 12 Component of HSE

effervescent base and tabletting lubricant; reduces alkalinity Ingredient Unit ~ By Function Reference (contd.) Weight Per (contd.) to Tablet Standards (contd. ) (contd. ) Sodium Carbonate 0.5 Component of HSE

(anhydrous) effervescent base and moisture stabilising agent A to aqueous solution of sodium dichloroisocyanurate has a pH
within the range of 5.5-7Ø The incorporation of an alkalinity reducing agent such as succinic acid ensures that the alkalinity, which is primarily bicarbonate alkalinity, is neutralised. This ensures that the nominal amount of the hypochlorous acid and/or the hypochlorite salt present in the sodium dichloroisocyanurate solution, is actually available.
The final pH will of course determine the relative amounts of hypochlorous acid and hypochlorite salt present.
The incorporation of adipic acid ensures a stable formula suitable for tabletting and the incorporation of the effervescent excipients ensure the effective and rapid release of hypochlorous acid and/or hypochlorite salt into solution, from the source.
It will be appreciated that the identity of the excipients can be changed or the excipients can be eliminated, depending on the final requirements or conditions under which the microbiocidal formulation of the present invention is to be used.

The microbiocidal formulation of the present invention will be equally suitable for use in powder, granulate or tablet form, each of which allows accurate quantification of the microbiocidally inactive source and of the excipient S quantities, in a given volume of water or any other suitable solvent. The studies set out in the remaining Examples were carried out with tablets of the microbiocidal formulation of the invention having the following composition:-Ingredient Weight (g/tablet) Succinic Acid 5.00 Sodium dichloroisocyanurate 2.21 Sodium Bicarbonate 1.036 Adipic Acid 1.13 Sodium Carbonate 0.044 Total Tablet Weight 9.42 The alkalinity reducing agent can be integrally incorporated in the microbiocidal formulation. Alternatively, the alkalinity reducing agent can be present in a coating on a powder, granulate or tablet embodiment of the microbiocidal formulation and, in that event, the alkalinity reducing agent is released into solution, to reduce the alkalinity level, before the microbiocidally active agent is released into solution. It will be appreciated that there is no need for a tabletting or lubricating agent in a powder or granulate formulation.
The desired pH of the final solution, in the case of sodium dichloroisocyanurate as the microbiocidally inactive source, is in the range 5.0-8.0, preferably 6.0-6.8.

The dissociation of hypochlorous acid into the hypochlorite ion is pH dependent. Thus, at pH 6.0, 97.30 of the hypachlorous acid is undissociated and, at pH 7.0, 78.10 of the hypochlorous acid is undissociated.
It is preferred that the anhydrous form of the microbiocidally inactive source be employed in the present microbiocidal formulations.
Exa~~r~ 1 a 2 The effect of varying water alkalinity on the pH of the microbiocidal formulation in the environment was investigated in the following manner:-Hard water, with a hardness of 342mg/1, is prepared by dissolving 304mg CaCl2 and 139mg MgC12.6H20 in 1 litre deionised water. Hard water, with an alkalinity of 100mg/l, 200mg/1 or 300mg/1 is prepared by dissolving 200mg, 400mg or 600mg NaHC03, with 304mg CaClz and 139mg MgC12.6Hz0 in 1 litre deionised water.
The microbiocidal formulation of the invention comprises one 9.428 tablet of Example 1 dissolved in sufficient hard water of 0, 100mg/1, 200mg/1 or 300mg/1 added alkalinity, to yield 25 ppm of available chlorine.
The comparative formulation has the following composition:

Ingredient Weight (o by weight per tablet) Sodium dichloroisocyanurate 50 Sodium Bicarbonate 22 Adipic Acid 24 Sodium Carbonate 4 One tablet (5g) of the comparative formulation is dissolved in sufficient hard water of 0, 100mg/1, 200mg/1 or 300mg/1 5 added alkalinity, to yield 25 ppm available chlorine.
In each case, the pH was measured at 10°C and the results are shown in Table 5.
Table 5: Effect of Alkalinity on pH
pH

Added Alkalinity Hard Water Comparative Formulation (ppm) (Diluent) Formulation of Invention (Dosed Water) (Dosed Water) 0 7.66 7.71 6.05 100 7.51 7.70 6.60 200 7.93 7.72 6.70 300 8.08 7.78 6.72 An impact of alkalinity on the dissociation of hypochlorous acid into hypochlorite and, therefore, on microbiocidal efficacy is suggested by the above-mentioned results. Thus, current experimental data generated in standard hard water are likely to be inaccurate in field conditions where alkalinity is often present.
It is clear that added alkalinity affects the pH of both the comparative formulation solution and the formulation of the invention solution. Specifically, when the added alkalinity is greater than 100mg/1, the pH of the comparative formulation solution is outside the desired 6.0-6.8 pH range.
In contrast, even when the added alkalinity is 300mg/1, the pH of the formulation of the invention solution is still within the desired 6.0-6.8 pH range.
The usual recommended method of carrying out the cleaning and disinfection of an automated milking system is known as cold circulation cleaning.
By this procedure, the lumen of the milking system is pre-rinsed at the end of the morning milking process, with cold water, followed by a wash sequence involving circulation of 45 litres of a detergent solution containing 0.5% (227g/45 litres (0.51b/10ga1)) of an approved caustic detergent, for 10 minutes, which detergent solution is then recovered in the wash trough for reuse in the second daily wash. It is suggested not to post-rinse the caustic solution residue from the lumen until immediately before the next milking, since it is believed that successful cleaning and microbiocidal action on the lumen of the milking system depends on prolonged contact of this caustic residue with the lumen surfaces. A
hot wash at regular intervals is an essential part of the routine to remove built up milk deposits, inter alia. In order to get optimum results from cold cleaning, the following instructions are widely acceptable:-Prescribed Routines:
A) Cold Circulation Cleaning Wash fetters and outside of clusters and attach them to the fetters. Rinse lumen of system with 14 litres (3 gal) cold water per cluster. Dissolve an approved caustic detergent in cold water at the rate of 227g/45 litres (0.51b/lOgal) allowing about 9 litres (2 gal) of solution per cluster.
Circulate the solution for 10 min having allowed the first 5 litres (1 gal) to run to waste. Return all the solution to the wash trough and retain for the second daily wash. Leave the clusters on fetters. Before next milking, rinse lumen of system with 14 litres (3 gal)~cold water per cluster to remove the caustic detergent residue. Add 28m1 (1 fl. oz) agricultural grade hypochlorite to the final ~7 litres (10 gal) of rinse water.
B) Regular hot wash (recommended at fortnightly intervals).
The data set out in Table 6 were obtained from two milking parlours on the same farm, one of which (control parlour) used the above-mentioned prescribed routine and the other of which (trial parlour) used the following alternative routine.

Wash fetters and outside of clusters and attach them to the fetters. Rinse the milking system through with 14 litres of cold clean water per cluster. Dissolve an approved caustic detergent in cold water at the rate of 2278/45 litres (0.51b/lOgal) allowing about 9 litres (2 gal) of solution per cluster. Make this cold wash up each day and use twice only.
Having allowed the first 5 litres (1 gal) to run to waste, circulate the solution for 10 minutes. Return all the remaining solution to the wash trough and retain for the second daily wash. Leave clusters on fetters. Next, post-rinse (at 2pm after morning use s2~ immediately after evening use) the system with 14 litres (3 gal) of cold clean water per cluster, to remove all traces of the caustic detergent residue. Prepare a microbiocidal formulation of the invention (dissolve two 9.428 tablets in about 11 water; the tablet comprises 5.Og succinic acid, 2.218 sodium dichloroisocyanurate, 1.0368 sodium bicarbonate, 1.138 adipic acid and 0.0448 sodium carbonate) and add same to 661 of the rinse water of the final rinse cycle. Suck through the milking system and allow to drain completely.
The trial was continued for two weeks. Table 6 gives the week 1 and week 2 results of the microbiological count of plant rinses after circulation during the trial.

TABLE 6: Microbiological count of plant rinses (count per ml of rinse water) Microbiological Week 1 Week 2 Count Control Trial Control Trial Total bacterial 1300 380 600 410 Count Psychotrophic 340 119 80 5 Thermoduric 3 1 4 5 The total bacterial counts and the psychotrophic counts for each of week 1 and week 2 show a superior performance for the microbiocidal formulation of the present invention. In addition, there was little evidence of a protein-like film build-up after two weeks use of the microbiocidal formulation of the invention.
A laboratory comparison was carried out to determine the microbiocidal efficacy of the test formulation as described in Example 1, of the comparative formulation (5g tablets) as described in Example 2 and of a sodium hypochlorite solution (Merck), supplied by Lennox Chemicals, Dublin, Ireland. The objective was to establish that the formulation of the present invention was more effective as a microbiocide than a conventional sodium hypochlorite solution and a conventional tableted formulation, as per Example 2, in "field" water.

The general format of the testing schedule is derived from BS3286:1960. The tests were carried out using 0.1% milk as an organic load. The milk used was unpasteurised milk with an approximate somatic cell count of 300 x 103. The test 5 organisms were Staphylococcus aureus, isolated from a clinical case of mastitis in dairy cows, and Bacillus subtilis, a control culture obtained from ATCC. Contact times for the three products were 5 mins, 10 mins, 6 hrs and 24 hrs .
The concentrations of the three products were 25 ppm (mg/1) of available chlorine. The tests were carried out at 10°C.
Dilutions in all cases were made in a diluent comprising water with an alkalinity of 300 mg/1 expressed as calcium carbonate (CaC03) equivalent and a hardness of 342mg/1 expressed as calcium carbonate (CaC03). The pH of the water was checked before and after addition of trial product.
Inactivation of the products under test was carried out by placing lml of reactant mix, after the appropriate contact time, in 9mls of sterile inactivation fluid. The inactivation will neutralise the effect of the disinfectant.
Dilutions were made at the contact times mentioned, and the inactivated fluids were cultured onto blood agar and MacConkey agar using standard laboratory practices.
Incubation was carried out for 18-24 hrs at 37°C. The inoculum consisted of 6mls of the test organism (1 x 104 orgs/ml) plus 4 mls of O.lo milk. The disinfectant dilution:inoculum ratio was 50:50. Controls substituted water, i.e. water with a hardness of 342mg/1 and an alkalinity of 300mg/1, instead of the trial product. The minimum level (pass criteria) that is required will be 99.99%
over viable count (cfu/ml).
The inactivation fluid comprises lecithin - soya (3g), Tween 80 (30m1), sodium thiosulphate (5g), L-histidine (lg), phosphate buffer (0.25N; 10m1), and purified water - made up to 1 litre. The inactivation fluid was sterilised at 121°C
for 15 minutes.
The "field" diluent comprises NaHC03 (600mg), CaClz (304mg) and MgC12.6Hz0 (1339mg), which is made up to 1,OOOml with deionised water.

RESULTS
Table 7 pH of "field"diluent . 7.98 pH of 25 ppm sodium hypochlorite solution . 8.21 pH of 25 ppm Agrisept Tabs solution . 7.73 pH of 25 ppm formulation of invention solution . 6.80 Organism: Bacillus subtilis Product (cfu) Time Sodium Agrisept Formulation of Hypochlorite Invention 5 mins >100 cfu* >100 cfu 59 mins >100 cfu >100 cfu 90 6 hrs >100 cfu >100 cfu 18 24 hrs >100 cfu 8 cfu N/G

N/G = No Growtri ~onLrol ~.vu~~~
*The indication of >100 cfu/ml means that the organisms were too numerous to quantify.

Organism: Staphylococcus aureus Product Time Sodium Agrisept~ Trial Product Hypochlorite mins >100 cfu N/G N/G

20 mins 88 cfu N/G N/G

6 hrs N/G N/G N/G

24 hrs N/G N/G N/G

5 N/G = No Growth Control Count 1 x 10°
A laboratory comparison of the efficiency of a test formulation as described in Example 1 against the conventional microbiocide, sodium hypochlorite, at two different concentrations, under control conditions. The objective was to establish that the formulation of the present invention was more effective as a microbiocide at a very low concentration, 25 ppm, when compared to sodium hypochlorite and that, even if the concentration was increased, by a factor of 10, to 250 ppm, the formulation of the present invention was much more efficient in "field"
water.
The testing procedure of Example 4 was followed, with the following amendments. The test organisms were Bacillus subtilis, Salmonella typhirnurium (phage type 104), Listeria monocytogenes and Clostridium butyricum.

The concentrations were 25 ppm and 250 ppm for each of hypochlorite and formulation of the present invention. The organic load was 0.1% milk (25 ppm) or 5o yeast (250 ppm).

Organism: Salmonella typhimurium phage type 104 25 ppm 250 ppm Time Control Test Control Test (Hypochlorite) Product (Hypochlorite) Product 5 mins 200-500 100-200 100-200 0 mins 100-200 100-200 100 0 30 mins 100-200 100 100-200 0 1 hour 100-200 100-200 100-200 0 6 hours 100-200 42 100 0 24 hours 100-200 100-200 100 0 10 Organism: Listeria monocytogenes 25 ppm 250 ppm Time Control Test Control Test (Hypochlorite) Product (Hypochlorite) Product 5 mins 100-200 100-200 91 0 10 mins 100-200 100-200 200-500 0 30 mins 100-500 100-200 200-500 0 1 hour 200-500 100-200 200-500 0 6 hours 100-200 100-200 100-200 0 24 hours 100-200 0 >200 100-200 Organism: Clostridium butyricum 25 ppm 250 ppm Time Control Test Control Test (Hypochlorite) Product (Hypochlorite) Product 5 mins 66 20 63 10 10 mins 48 32 41 5 30 mins 34 26 61 6 1 hour 33 9 40 3 6 hours 13 18 13 4 24 hours 0 0 0 0 Organism: Bacillus subtilis 25 ppm 250 ppm Time Control Test Control Test (Hypochlorite) Product (Hypochlorite) Product 5 mins 28 23 27 10 10 mins 31 28 26 13 30 mins 21 23 35 4 1 hour 21 12 28 4 6 hours 26 32 23 1 24 hours 15 28 22 0 These data demonstrate the difference between sodium 10 hypochlorite and the formulation of the present invention.
The trial was carried out simultaneously on both products at two different concentrations, 25 ppm and 250 ppm. The results of the trial clearly demonstrate that, at 250 ppm, the formulation of the present invention is microbiocidally superior to the conventional hypochlorite. At 25 ppm, the difference is smaller but, in most cases, the formulation of the present invention is still superior.
Formulation (A) Total Formulated Weight.....3.2 grams Composition: Sod. Dichloroisocyanurate... 25% (w/w).....800mg Sod. Bicarbonate............ 350 (w/w)....1120mg Sod. Carbonate.............. .5% (w/w).....160mg Succinic Acid...............35% (w/w)....1120mg Formulation (B) Total Formulated Weight.....5.56 grams Composition: Sod. Dichloroisocyanurate...14.39% (w/w)...800mg Sod. Bicarbonate............20.14% (w/w)..1120mg Sod. Carbonate..............-2.880 (w/w)...160mg Succinic Acid...............62.59% (w/w)..3480mg The general format of the testing schedule is derived from BS3286:1960 and the testing procedure of Example 4 was followed, with the following amendments. The tests were carried out using 0.1% milk as an organic load. The test organism was Bacillus subtilis BGA, a control culture obtained from ATCC. The contact time for each of the formulations was 5 mins, 10 mins, 1 hour and 6 hours. The concentration of each formulation was 100 ppm (mg/1) available chlorine in a volume of 5 litres of diluent water.
All tests were carried out at 10°C. The diluent water had a total alkalinity of 400mg/1 expressed as calcium carbonate (CaC03) equivalent and a hardness of 342mg/1 expressed as calcium carbonate (CaC03). The pH of the water was measured before and after the addition of the various formulations.
The inactivated fluids were cultured onto Colombia Blood Agar using standard laboratory practices. Incubation was for 18 hours at 37°C. The concentration of the inoculum was 1.25 x 106 orgs/ml. The "field" diluent comprises NaHC03 (750mg), CaCl2 (304mg) and MgC12.6Hz0 (1339mg), which is made up to 1,OOOm1 with deionised water.
Table 14 pH of "field" diluent: 8.01 pH of 100 ppm solution of test formulation A (3.2g): 6.68 pH of 100 ppm solution of test formulation B (5.52g): 5.41 Table 15 Contact Time Test Formulation A Test Formulation B

5 mins >100 cfu* 89 10 mins >100 cfu 52 1 hour >100 cfu 64 6 hours >100 cfu 91 *The indication of >100 cfu/ml means that the organisms were too numerous to quantify.
The results show that Test Formulation B is superior to Test Formulation A in suppressing growth of Bacillus subtilis BGA
under the conditions described. The residual resistance of the organism is due to various factors limiting the formulation's microbiocidal activity, specifically, its spore form, the high concentration of the inoculum organism used and the high total alkalinity of the diluent.
It will be appreciated that the microbiocidal formulation of the present invention has applicability in a milking apparatus at the end of a post-milking apparatus washing procedure (as exemplified in Example 3); in any pre-pasteurisation holding system; and in any post-pasteurisation apparatus against post-pasteurisation contaminant micro-organisms.
Although the above-mentioned results concern sterilisation of the lumen of a milking apparatus, it is expected that the microbiocidal formulations of the present invention will be particularly suited to the treatment of water, where the world Health Organisation (WHO) have now set the limit for residual chlorine in treated water at only 5mg/1. Thus, with conventional microbiocidal formulations, such water is being inadequately treated, since at least some of the hypohalous acid/hypohalite salt is being neutralised by the alkalinity present and, if excess conventional microbiocidal formulation is provided, then, in areas of low alkalinity, the WHO limits may be exceeded.
It will be appreciated, therefore, that the microbiocidal formulations of the present invention provide effective water treatment by giving effective microbiocidal effect without exceeding the WHO recommended limits.

It will also be appreciated that the microbiocidal formulation of the present invention, although exemplified in respect of a milking apparatus, has general applicability in any apparatus used in the production, preparation or processing of food or beverages and, indeed, in the treatment of water for human or animal consumption or of process liquids.
Sodiumdichloroisocyanurate - 2.2108 Adipicacid - 1.1308 Sodiumbicarbonate - 1.0368 Sodiumcarbonate - 0.0448 Succinic - 0.5618 acid A microbiocidal formulation of the above-mentioned composition is useful, following dissolution in five litres of a diluent having a pH of 7.5, a total alkalinity of 100mg/1 as calcium carbonate and a total carbonic carbon of 2.lmmol/1 (calculated) so as to achieve, following dissolution in the diluent, a pH of 6.0 and a FAC to total Cl ratio of 1Ø
2 0 ~x~ l~e-8 Sodiumdichloroisocyanurate - 2.2108 Adipicacid - 1.1308 Sodiumbicarbonate - 1.0368 Sodiumcarbonate - 0.0448 Succinic - 2.6198 acid A microbiocidal formulation of the above-mentioned composition is useful, following dissolution in five litres of a diluent having a pH of 7.5, a total alkalinity of 400mg/1 as calcium carbonate and a total carbonic carbon of 5 8.5mmo1/1 (calculated) so as to achieve, following dissolution in the diluent, a pH of 6.0 and a FAC to total C1 ratio of 1Ø
Sodium dichloroisocyanurate - 2.210g Adipic acid - 1.1308 Sodium bicarbonate - 1.0368 Sodium carbonate - 0.0448 Succinic - 0.2188 acid A microbiocidal formulation of the above-mentioned composition is useful, following dissolution in five litres of a diluent having a pH of 7.5, a total alkalinity of 50mg/1 as calcium carbonate and a total carbonic carbon of l.lmmol/1 (calculated) so as to achieve, following dissolution in the diluent, a pH of 6.0 and a FAC to total C1 ratio of 1Ø
Sodium dichloroisocyanurate - 2.2108 Adipic acid - 1.1308 Sodium bicarbonate - 1.0368 Sodium carbonate - 0.0448 Succinic - 13.5898 acid A microbiocidal formulation of the above-mentioned composition is useful, following dissolution in twenty-five litres of a diluent having a pH of 7.5, a total alkalinity of 400mg/1 as calcium carbonate and a total carbonic carbon of 8.5mmol/1 (calculated) so as to achieve, following dissolution in the diluent, a pH of 6.0 and a FAC to total C1 ratio of 1Ø
Sodiumdichloroisocyanurate - 2.210g Adipicacid - 1.130g Sodiumbicarbonate - 1.0368 Sodiumcarbonate - 0.0448 Succinic - 3.3038 acid A microbiocidal formulation of the above-mentioned composition is useful, following dissolution in twenty-five litres of a diluent having a pH of 7.5, a total alkalinity of 100mg/1 as calcium carbonate and a total carbonic carbon of 2.lmmol/1 (calculated) so as to achieve, following dissolution in the diluent, a pH of 6.0 and a FAC to total Cl ratio of 1Ø
Ex~~le 12 Sodiumdichloroisocyanurate - 2.2108 Adipicacid - 1.1308 Sodiumbicarbonate - 1.0368 Sodiumcarbonate - 0.0448 Succinic - 0.4288 acid A microbiocidal formulation of the above-mentioned composition is useful, following dissolution in one litres of a diluent having a pH of 7.5, a total alkalinity of 400mg/1 as calcium carbonate and a total carbonic carbon of 8.5mmo1/1 (calculated) so as to achieve, following dissolution in the diluent, a pH of 6.0 and a FAC to total C1 ratio of 1Ø
Sodium dichloroisocyanurate - 2.2108 Adipic acid - 1.1308 Sodium bicarbonate - 1.0368 Sodium carbonate - 0.0448 Succinic - 0.0178 acid A microbiocidal formulation of the above-mentioned composition is useful, following dissolution in one litres of a diluent having a pH of 7.5, a total alkalinity of 100mg/1 as calcium carbonate and a total carbonic carbon of 2.lmmol/1 (calculated) so as to achieve, following dissolution in the diluent, a pH of 6.0 and a FAC to total C1 ratio of 1Ø
Referring to Examples 7-13, Example 13 is an example of a microbiocidal formulation, in which the total alkalinity of the diluent water plays a very minor role, in that it is necessary to merely add 0.0178 succinic acid to the adipic acid already present in the microbiocidal formulation, in order to achieve a target dosed pH of 6Ø
Concerning Examples 7, 9 and 12, the total alkalinity of the diluent water plays a minor role, in that it is necessary to add 0.2-0.6, preferably, 0.218-0.5618 succinic acid to the adipic acid already present in the microbiocidal formulation, in order to achieve a target dosed pH of 6Ø
Concerning Example 8, the total alkalinity of the diluent water plays a medium role, in that it is necessary to add 2.6198 succinic acid to the adipic acid already present in the microbiocidal formulation, in order to achieve a target dosed pH of 6Ø
Concerning Examples 10 and 11, the total alkalinity of the diluent water plays a major role, in that it is necessary to add 3-158, preferably, 3.303-13.5898 succinic acid to the adipic acid already present in the microbiocidal formulation, in order to achieve a target dosed pH of 6Ø

Claims (12)

CLAIMS:

1. A microbiocidal formulation suitable, following dissolution in a diluent to form a microbiocidal solution for the microbiocidal treatment of an environment, the environment being selected from the group consisting of an external surface or a lumen of an apparatus used in the production, preparation or processing of food or beverages, process liquid and liquid for human or animal consumption, the formulation comprising:
sufficient diluent alkalinity neutralising agent so that, following dissolution in the diluent, an alkalinity of no more than 100 mg/l, preferably no more than 50mg/l bicarbonate alkalinity is observed in the microbiocidal solution, the diluent alkalinity neutralising agent being a comestibly acceptable acid or its salt or a mixture thereof;
sufficient pH neutralising agent so that, following dissolution in the diluent, a pH of 5.0-8.0, preferably 6.0-6.8, is observed in the microbiocidal solution; and a microbiocidally effective amount of an alkalinity sensitive microbiocidal agent, the microbiocidal agent being available halogen released, in aqueous solution, from a halogenated isocyanuric compound or a salt thereof, selected from the group consisting of sodium dihaloisocyanurate, potassium dihaloisocyanurate and trihaloisocyanuric acid;
the formulation being adapted to release a microbiocidally effective amount of the available halogen over a microbiocidally effective period of time.

2. A microbiocidal formulation according to claim 1, in which the halogenated isocyanuric compound is sodium dichloroisocyanurate.

3. A microbiocidal formulation according to claim 1 or 2, in which the microbiocidally effective amount of available halogen is released from 1 to 5000 ppm of the halogenated isocyanuric compound or a salt thereof and the microbiocidally effective period of time is in the range of 10 seconds to 48 hours.

4. A microbiocidal formulation according to any one of the preceding claims, which is effective against E.coli or Pseudomonas or is a resistant micro-organism, still more preferably a thermoduric or thermophilic organism, most preferably a species of micro-organism selected from Bacillus, Micrococcus, Microbacterium, Clostridium, Listeria, Alcaliigenes, Arthrobacter, Lactobacillus, Serratia or any other spore forming species.

5. A microbiocidal formulation according to any one of the preceding claims, in which the diluent alkalinity neutralising agent is succinic acid or a salt thereof.

6. A microbiocidal formulation according to any one of claims 1 - 5, in which the diluent alkalinity neutralising agent is citric acid or a salt thereof.

7. A microbiocidal formulation according to any one of the preceding claims, suitable for dissolution in the diluent, the diluent containing, in total, 2g alkalinity as calcium carbonate, in which the diluent alkalinity neutralising agent comprises 53.3% (by weight) of the microbiocidal formulation.

8. A microbiocidal formulation according to any one of claims 1-6, suitable for dissolution in the diluent, the diluent containing, in total, 10g alkalinity as calcium carbonate, in which the diluent alkalinity neutralising agent comprises 55.7-83.1%, preferably 57.4-81.7% (by weight) of the microbiocidal formulation.

9. A microbiocidal formulation suitable, following dissolution in a diluent to form a microbiocidal solution for the microbiocidal treatment of an environment, the environment being selected from the group consisting of an external surface or a lumen of an apparatus used in the production, preparation or processing of food or beverages, process liquid and liquid for human or animal consumption, the formulation comprising:
sufficient diluent alkalinity neutralising agent so that, following dissolution in the diluent, an alkalinity of no more than 100 mg/l, preferably no more than 50mg/1 bicarbonate alkalinity is observed in the microbiocidal solution, the diluent alkalinity neutralising agent being a comestibly acceptable acid or its salt or a mixture thereof;
sufficient pH neutralising agent so that, following dissolution in the diluent, a pH of 5.0-8.0, preferably 6.0-6.8, is observed in the microbiocidal solution; and a microbiocidally effective amount of an alkalinity sensitive microbiocidal agent, the microbiocidal agent being available halogen released, in aqueous solution, from a halogenated isocyanuric compound or a salt thereof, selected from the group consisting of sodium dihaloisocyanurate, potassium dihaloisocyanurate and trihaloisocyanuric acid;
a lubricant;
the formulation being adapted to release a microbiocidally effective amount of the available halogen over a microbiocidally effective period of time, with the proviso that more than 35% (by weight) of an organic acid is present as the diluent alkalinity neutralising agent.

10. A method for optimising the microbiocidal effectiveness of a microbiocidal formulation, the formulation for dissolution, in use, in a diluent to form a microbiocidal solution, the method comprising providing sufficient diluent alkalinity neutralising agent in the microbiocidal formulation so as to observe, following dissolution in the diluent, an alkalinity of no more than 100 mg/l, preferably no more than 50mg/l bicarbonate alkalinity in the microbiocidal solution.

11. A microbiocidal solution comprising:
a microbiocidal formulation in powder, granulate or tablet form, dissolved in a diluent, the solution having no more than 100 mg/l, preferably no more than 50mg/l bicarbonate alkalinity;
a pH of 5.0-8.0, preferably 6.0-6.8; and a microbiocidally effective amount of an alkalinity sensitive microbiocidal agent being available halogen released, in use, from a halogenated isocyanuric compound or a salt thereof; selected from the group consisting of sodium dihaloisocyanurate, potassium dihaloisocyanurate and trihaloisocyanuric acid;
the solution being adapted to release the microbiocidally effective amount of the alkalinity sensitive microbiocidal agent over a microbiocidally effective period of time.

12. A microbiocidal formulation according to claim 1 or 9, in which the diluent alkalinity neutralising agent is a comestibly acceptable organic acid or its salt or a mixture thereof.