This invention relates to the hygienic treatment of water.

There are a number of applications where it is desirable to treat water so as to counteract any microbial contaminants therein. Frequently it is adequate to kill any such microbial contaminants.

A known technique for doing this is to liberate silver ions into the water, by an electrochemical reaction. Silver ions are known to have an antimicrobial action. This use of electrochemically generated silver ions has been discussed in a number of literature articles including

Spadaro et al Antimicrobial Agents and Chemotherapy _, 637 - 642, (1974) .

Landeen et al Water Sci Tech 2JL, 267-70, (1989) Yahya et al Can. J. Microbiol 3 109-116, (1990)

Pyle et al J. Applied Bacteriology 72, 71-9 (1992)

UK Patents 1507324 and 1512146.

The silver is normally liberated into a solution at a concentration which does not exceed 0.1 parts per million (ppm) . It may be noted that standards for potable water in UK and USA impose limits of 0.08 and 0.05 ppm respectively. At such concentrations, silver is rather slow in its cell killing action.

Suppliers of equipment for the electrolytic liberation of silver ions into water are Tarn-Pure Limited, High Wycombe, England and Tarn-Pure USA, Las Vegas, Nevada, USA. As mentioned in the Yahya et al and Pyle et al articles, copper and silver ions may be generated together, using an anode which contains both metals.

We have now found that silver can be rendered more effective or more stable or both by including in the water a material which is able to form a co-ordination complex with the silver ions.

Accordingly the present invention provides a method of killing microbial contaminants in water by introducing silver ions into solution in the water characterised by including in the water an organic ligand which is able to form a soluble co-ordination complex with the silver ions, especially an amphoteric or zwitterionic surfactant, a polyether, or a polycarboxylate.

The invention is particularly applicable when the silver ions are liberated into solution by the known step of electrochemical reaction, although the silver could conceivably be introduced into the water in some other way, such as by adding a solution of a silver salt.

The electrolytic liberation of silver ions requires apparatus which provides an electrolytic cell, with a silver-containing anode in contact with water to be treated. This water may be in contact also with the cathode, or the cathode may be located in different electrolyte separated by a permeable membrane from the water to be treated.

The anode may be silver, or a silver alloy from which both silver ions and other ions are liberated.

Various metals may be used as the cathode. Stainless steel (which is an alloy principally containing iron, chromium and nickel) is satisfactory. Silver itself may usefully be used as the cathode, and the direction of the current may be reversed periodically, so that both electrodes are consumed.

The water to be treated needs some conductivity. The concentration of ions which occur in normal water supplies is usually sufficient for this. If required, a small quantity of an electrolyte may be added to the water.

A considerable number of organic chemicals can serve as ligands able to form water soluble complexes with silver. Such ligands should have a functional group capable of taking a negative charge for co-ordinating to silver. These groups will generally contain such heteroatoms as oxygen, sulphur and phosphorus. Examples of functional groups which bind to silver are thioethers, ethers, thiocarbonates, amines, imines, pyrazoles, benzimidazoles and phosphines. The ligands do not need to carry an overall negative charge. For instance zwitterionic molecules can serve as ligands.

One preferred category of material which is useful for forming co-ordination complexes is polycarboxylates, especially polycarboxylates wherein each ligand molecule contains at least three carboxylate groups able to co¬ ordinate to silver. Particularly suitable are oligomers and polymers bearing carboxylate groups such that a ligand molecule bears at least three carboxylate groups capable of co-ordinating to silver.

Such ligands display a high affinity for silver and can enhance the stability of the silver ions against precipitation by hydroxyl or chloride ions. Surprisingly, however, the coordination complexes of silver with such ligands also display good antimicrobial/biocidal activity - which would not be expected when the silver is strongly bound by the ligand.

Preferably such ligands contain an average of more than one carboxylate group per monomer residue in the oligomer or polymer. Such ligands may contain monomer residues

which each contribute a plurality of carboxylate groups. Examples of such monomers are maleate, fumarate, itaconate, and aconitate.

It is preferred that the oligomers and polymers contain a majority of monomer residues bearing carboxylate groups, especially a majority of monomer residues which, as discussed, each contain a plurality of carboxylate groups.

A polycarboxylate will, usually, be made from olefinically unsaturated monomers. It may be a random or block copolymer. One or more monomers which bear a plurality of carboxylate groups, such as maleate, may be copolymerised with monomers which bear a single carboxylate group, such as acrylate or methacrylate. Other possible comonomers do not have ionisable carboxylate groups, as for example vinyl acetate.

A preferred category of monomers, which may provide 20. mole% or more, especially 50 mole% or more, of a polymer or oligomer, have the formula

R- CH C0 M. / 2 2 1

C = C

^R 2 ^CH 2 ^C °2 ^M 2

wherein each of R and R_, which may be the same or different, represents a hydrogen atom, a methyl group or an ethyl group, and each of M and M , which may be the same or different, is a hydrogen atom or a solubilising cation.

Itaconate is an example of such a monomer. Such monomers may be used to provide 20 to 95 mole % of a polymer or oligomer in which the residues of comonomer have the

formula

-CH ₂ -CH-

OR ₃

wherein R- represents a hydrogen atom or an acyl group -COR. in which R is a C. to C alkyl group.

Polymeric ligands can be prepared by conventional polymerisation techniques. For example a procedure for the polymerisation of maleic anhydride is given by Lang et al in J. Polymer Sci . issue 162, page 532 (1961) . The resulting polymer can readily be hydrolysed to polymaleate by treatment with aqueous sodium hydroxide.

In general, olefinically unsaturated acids and diacids can be polymerised alone or jointly with other olefinic monomers such as vinyl acetate by conventional techniques for radical polymerisation.

Polyitaconate homopolymers are described in US 3055 873 and 3405060. Copolymers with acrylic acid are disclosed in EP 506 246. Other polycarboxylates are described in EP 193 360 and US 4725655.

Polycarboxylates can also be obtained from monomers which do not contain olefinic unsaturation but instead undergo condensation polymerisation. Examples are malonate, isocitrate, citrate, succinate, tartrate, oxaloacetate, methylmalonate, carballylate, aspartate, glutamate and gamma-carboxyglutamate.

Another type of material which is useful as the organic ligand is amphoteric surfactants containing amino groups and carboxylate groups. Preferred among these are amphoteric surfactants containing at least two secondary amino groups as well as a carboxylate group.

Amphoteric surfactants will also generally incorporate an alkyl or alkenyl chain of 7 to 18 carbon atoms.

Yet another type of material which may be used as the organic ligand is a betaine notably amido betaines of formula:

R 4.-CONH- (CH )

In both formulae R. is a C„ to C 8 straight or branched alkyl or alkenyl group; R.. and R, are each C to C_ alk_ or C to C- hydroxyalkyl. n is 2 to 4 especially 3.

Corresponding sulphobetaines have the above formula with -CH ₂ CO ^" replaced by

A further category of materials useful as organic ligands are a group of naturally occurring antibiotics known as polyether ionophores. These molecules incorporate a number of oxygen atoms, frequently in furan and pyran rings so that they are polyethers. They are known to have cation complexing properties. A review and listing of such materials is provided in "Polyester antibiotics" edited by J.W. Westley.

Ideally water treatment in accordance with this invention is carried out using the organic ligand and silver ions in such quantities that complexing of the silver is not restricted by shortage of ligand.

In practice some surplus of ligand will usually be harmless, while the benefit of the invention will be obtained in part if there is some surplus of silver ions in solution. Consequently it is preferred that substantially all the silver is in the form of a complex, and is accompanied by surplus ligand.

Usually the mole ratio of silver to ligand will be from 10:1 up to 1:100. Preferably there will be enough ligand to provide the stoichiometric equivalent of the concentration of silver ions so that the silver to ligand mole ratio lies in a range from 1:1 to 1:100. Frequently the concentration of organic ligand will lie in a range from 0.001 or 0.005 to 3,000 ppm, especially 0.5 or lppm up to 500 ppm. Concentrations of ligand higher than 3,000 ppm may be employed if desired.

The concentration of silver which is maintained in the water will frequently lie in a broad range from 0.001 to 1000 ppm, especially 0.01 or 0.1 to 100 ppm. The concentration more preferably does not exceed 25 ppm.

If the invention is applied to the treatment of a body of water which is kept for a long time - notably a body of water which is kept in circulation, such as the water of a swimming pool or an air conditioning system, a single addition of ligand to the water may suffice for a long period.

On the other hand if the water is being consumed and replaced, the organic ligand will have to be added to it regularly or continuously, and the introduction of silver ions, preferably by electrolytic liberation, will need to be regular or continuous.

Example 1 .

Silver ions were generated electrolytically in water, and in aqueous solutions of an organic ligand. A control has water with neither silver nor organic ligand present. The water, which was also used for the preparation of the ligand solutions, was sterile distilled water to which had been added a very small crystal of sodium sulphate to confer conductivity. The ligand was a commercial mixture of amphoteric surfactants of formulae C ₁₂ H ₂₅ -NH- (CH ₂ ) ₃ -NHCH ₂ C0 ₂ H and C ₁₂ H ₂₅ -NH- (CH ₂ ) -NH- (CH ₂ ) -NHCH ₂ C0 ₂ H available as Amphobac 4 from Lonza Inc. It was used at concentrations of 2ppm and 20ppm.

Silver was generated electrolytically, using a pair of silver wires dipping into the water or ligand solution as electrodes. These were connected to a constant current direct current supply, for sufficient time to liberate 3 ppm silver ions into solution.

10ml volumes of each solution were then inoculated with 0-1 ml of an aqueous suspension containing five species of microorganism, and thoroughly mixed. The five species were four bacteria, namely Staphylococcus aureas, Streptococcus faecalis, Pseudomonas aeruginosa and Proteus mirabilis and a yeast, Saccharomyces cerevisiae.

After a period of 5 minutes a 1 ml portion was removed and transferred to a quenching solution containing sufficient sodium thiosulphate to precipitate the silver ions . In a separate test, the effect of sodium thiosulphate on the microorganisms was checked: it was found that it did not reduce their viability.

The quantities of surviving micro organisms were determined by serial dilution, spreading the diluted

solution on an agar plate, incubating and counting the number of colonies of each species. From this count, and the extent of dilution, the concentration of surviving micro organisms in the test solution was calculated. The results are set out in the following table and are given as the logarithm of the number of survivors .

TABLE 1

As can be seen from the above table, electrochemically liberated silver reduces the microorganism concentrations by three or four orders of magnitude, but a considerably greater reduction is achieved when the organic ligand is also present.

Example 2

The procedure of Example 1 was repeated, using Lasalocid as organic ligand, at a concentration of 15ppm. Lasalocid is a polyether antibiotic of formula

Results are given in Table 2 below.

TABLE 2

Example 3

The procedure of Example 1 was repeated using a longer contact time, using E.coli and Staph aureus as the microorganism species, and using an amidopropyl betaine as ligand. This had the formula

CH,

C ₁₃ H ₂₇ -CO-NH- (CH ₂ ) ₃ N ⁺ -CH ₂ C0 ₂ ^"

CH ₃

and was used at a concentration of 10 ppm so as to provide an excess of ligand over silver. It is available as Tegobetaine from Goldschmidt .

The silver concentration was 2 ppm.

The results obtained were:

log ₁₀ surviving cells after 1 hour

E.coli St. aureus water 7.07 6.49

Tegobetaine 6 . 36 3 .47 Silver 2 . 61 2 . 91

Silver + Tegobetaine <1 1 . 60

Example 4

A copolymer was prepared from 20 mole % vinyl acetate and

80 mole % itaconic acid.

This polymer had the formula

-CH-

OR, n m

wherein R_ indicates an acetyl group, and the indices n and m indicate the numbers of repeating units.

Vinyl acetate (9.4ml, 0.1 mole) , itaconic acid (48g, 0.4 mol) and degassed water (200 ml) were charged into a flange flask and stirred at 40°C under a nitrogen atmosphere. The redox initiator, comprising sodium persulphate (0.8g) and sodium metabisulphite (0.4g) , was added in three increments each of 0.3g over four days. The polymer-containing solution was concentrated to approximately half volume using a rotary evaporator and the concentrate poured into acetone. This precipitated the polymer which was washed with portions of acetone. A small sample was taken for analysis and molecular weight determination. The copolymer was then dissolved in water, neutralised with 6% sodium hydroxide solution to a pH of 8.5 and freeze dried.

The procedure of Example 3 was repeated using the copolymer as the ligand, at a concentration of 16 ppm, again providing an excess of ligand over silver.

The results obtained were log ₁₀ surviving cells after 1 hour

E.coli St. aureus water 7.07 6.49

Copolymer 6.36 5.49

Silver 2.61 2.91

Silver + Copolymer <1 3.07

It can be seen that the copolymer enhanced the activity against E.coli without significant deteriment to the activity against St. aureus .

Example 5 lOOg of the polyitaconate-vinyl acetate copolymer in sodium salt form, as in the previous Example, was dissolved in 500ml water. Silver nitrate (30g) dissolved in 80ml water was added over about 5 minutes. The whole mixture was stirred for twenty minutes and then ethanol (1.5 litre) was added. A fine white precipitate formed, which was filtered off and dried.

Analysis by H n.m.r in deuterium oxide confirmed presence of the polyitaconate-vinyl acetate copolymer. Silver analysis by silver selective electrode showed 15% silver by weight. Analysis was confirmed by atomic absorption spectroscopy.

0.25g of the above complex was dissolved in 250ml dilute acid to yield a solution containing 1000 ppm of the complex. Sodium hydroxide solution was slowly added. Measurements were made of pH and simultaneous measurements were made of free silver ion concentration. At pH 5 all the silver ions were in solution. Increasing alkalinity reduced the concentration of free silver as ionisation of the polymer enabled it to complex with silver ions. The pH was progressively increased to pH 10.5 without precipitation occurring. This absence of precipitation was shown by the optical transmission, which remained constant.

By contrast, a solution of silver nitrate (11 ppm) was rendered alkaline. At pH 8.0 or greater a brown precipitate of silver oxide was observed.

Example 6

The stability of the above complex towards chloride ion was compared with the stability of silver nitrate.

To 25ml of an aqueous solution containing 11 ppm silver

—6 nitrate (1.63 x 10 moles) was added 1ml of 0.005M

—6 potassium chloride solution (i.e. 5 x 10 moles) . The concentration of free silver ion dropped to <0.3 ppm

(limit of detection by apparatus) due to precipitation of insoluble silver chloride. The optical transmission of the solution dropped immediately from 100% to 70% relative to distilled water due to the formation of a silver chloride precipitate.

A sample of silver polyitaconate-vinyl acetate complex, prepared in Example 5, (0.133g) was dissolved in 25ml water. This contained about 1.8 x 10 -4 gram atoms of silver and represents a total silver concentration of about 800 ppm. However, the measured free silver ion concentration was only 5.2 ppm. To this solution was added 1ml of 0.005M potassium chloride solution (i.e. 5 x 10 ^~ moles) . The concentration of free silver ion dropped to 4.5 ppm. The optical transmission of the solution changed from 99% to 99.5% relative to distilled water. No insoluble particles were observed.

A further sample of the same complex (16.6 mg) was dissolved in 25ml water, made alkaline to pH 10 with

-6 sodium hydroxide solution. This was about 10 gram atoms of silver and a silver concentration of lOOppm.

An excess of potassium chloride solution was added (1ml of 0.1M solution) = 10 -4 moles. There was no visible formation of precipitate during a period of thirty minutes.

Example 7

An aqueous suspension was prepared containing Candida parapsilopsis (yeast) , Penicillium and Aspergillus spores (mould) Escherichia coli (Gram negative bacterium) and Staphylococcus aureus (Gram positive bacterium) .

lml of this suspension was mixed with 9ml of an aqueous solution of the silver complex of Example 1, containing 10 ppm silver. As a comparison a similar portion was mixed with an aqueous solution of the ligand only. As a control, a third portion was treated with water only.

After 30 minutes the numbers of surviving organisms were determined by serial dilution, plating on agar growth media and counting viable colonies.

The results obtained were as set out in the following table which shows that the ligand alone had no antimicrobial action whereas the silver complex was highly effective.

TREATMENT LOG ₁₀ SURVIVORS AFTER 30 MINS CONTACT CANDIDA MOULDS E.COLI S. AUREUS

ITACONATE 5.61 6.14 7.54 7.68 COPOLYMER ITACONATE <1 1.60 <1 <1 POLYMER + Ag

WATER 5.69 5.47 7.49 7.25