GB2103928A

GB2103928A - Control of industrial spoilage with nitroimidazole compounds

Info

Publication number: GB2103928A
Application number: GB08223894A
Authority: GB
Inventors: James Anderson Mcfadzean
Original assignee: May and Baker Ltd
Current assignee: May and Baker Ltd
Priority date: 1981-08-21
Filing date: 1982-08-19
Publication date: 1983-03-02
Also published as: JPS5839605A; CH655011A5; FR2517969B1; NL193053C; IE822000L; NL193053B; LU84345A1; SU1266473A3; DE3230887A1; AU8740882A; CA1192001A; IT8222901A0; KR840000875A; DK164599B; GB2103928B; AR229535A1; IT1152376B; ZA826043B; DE3230887C2; DK164599C

Abstract

Dimetridazole, metronidazole, secnidazole and ipronidazole are used to combat industrial spoilage of liquid organic materials containing water or aqueous liquids containing organic materials caused by obligate anaerobic sulphate-reducing bacteria.

Description

SPECIFICATION Control of industrial spoilage with nitroimidazole compounds This invention relates to a method of combatting industrial spoilage caused by obligate anaerobic sulphate-reducing bacteria and to compositions for use in the method.

Industrial spoilage of liquid organic materials and aqueous liquids containing organic materials, caused by the growth of microorganisms, which metabolize organic material with the concurrent formation of deleterious metabolites, is a widespread problem in many branches of industry. This growth of microorganisms requires the presence of free water in liquid organic material, e.g. an oil, since although the microorganisms can often pass into the organic, e.g. oil, phase, their growth and activity are largely confined to the water phase. It is, in practice, virtually impossible to exclude from liquid organic material the small initial amounts of water required for the commencement of growth of microorganisms and, indeed, water, in larger or smaller amounts, is deliberately incorporated into many liquid organic materials used in industry.A wide variety of bacteria, moulds and yeast are encountered in spoilage of liquid organic material and it is equally virtually impossible to exclude all such microorganisms from the liquid organic materials. Examples of such liquid organic materials and aqueous liquids containing organic materials which are subject to industrial spoilage caused by the growth of microorganisms include petroleum hydrocarbon and vegetable oils and liquid products containing them used in industry, including fuel oils, lubricating oils and mineral oil-based hydraulic fluids, oil-in-water emulsions, e.g. metal-working fluids, water-in-oil emulsions, for example water-in-oil emulsion hydraulic fluids and aqueous solutions and emulsions of glycols used as metal-working fluids.

Other aqueous liquids containing organic materials which are subject to serious spoilage caused by the growth of microorganisms are water-based paints, paper-pulp sulphite processing liquids, and even fluids which are largely inorganic, for example process waters, electro-plating solutions and oil-well injection water.Industrial spoilage of liquid organic materials and aqueous liquids containing organic materials, e.g. petroleum hydrocarbon and vegetable oils, caused by the growth of microorganisms can result in the corrosion of metal storage tanks, for example in the storage of fuel oils and in the extraction and processing of mineral oils, and of metal work pieces, for example in the machining, drilling, grinding and rolling of metals, when spoilage occurs in metal-working fluids used as lubricants and coolants during the working of metals, in the breakdown of additives in lubricating oils with consequent deterioration of the lubricating properties of the oils, in the reduction of sulphonate and sulphate emulsifiers and the oxidation or reduction of nitrogen-containing emulsifiers and corrosion inhibitors with consequent deterioration and instability of oil-in-water and water-in-oil emulsions, in the reduction of oil-water interfacial tension in stored fuel oil with consequent failure of oil-water separation and contamination of the fuel oil by the water, and in blockage of pipes, filters and orifices by the bodies of microorganisms.

The oxygen levels in liquid organic materials and aqueous liquids containing organic materials, e.g.

petroleum hydrocarbon and vegetable oils and liquid products containing them, used in industry are frequently low and may be further reduced by the growth of aerobic and facultative anaerobic microorganisms, thereby favouring the growth of obligate anaerobic sulphate-reducing bacteria (By the term 'obligate anaerobic sulphate reducing bacteria' as used in the present specification is meant obligate anaerobic bacteria which are able to abstract metabolically oxygen from compounds containing sulphur in a state of oxidation and leave it in a reduced state, usually hydrogen sulphide, or metabolically reduce elemental sulphur to hydrogen sulphide).Examples of obligate anaerobic bacteria which cause industrial spoilage include Desulfovibrio desulfuricans, Desulfovibrio gigas and Desulfotomaculum nigrificans (Clostridium nigrificans or Desulfovibrio orients). Obligate anaerobic sulphate-reducing bacteria metabolise reductively sulphates and sulphonates, for example sulphate and sulphonate-containing emulsifiers, e.g. alkyl sulphates and alkyl aryl sulphonate emulsifiers, and sulphurized oils and fats and free sulphate ions present in water, with the liberation of hydrogen sulphide, and result in particularly severe problems of industrial spoilage in liquid organic materials and aqueous liquids containing organic materials, for example petroleum hydrocarbon and vegetable oils, e.g. fuel oils and lubricating oils, mineral oil-based hydraulic fluids, oil-in-water emulsions, e.g. metal working fluids, water-in-oil emulsions, e.g. water-in-oil emulsion hydraulic fluids, and aqueous solutions and emulsions of glycols used as metal-working fluids, with the production of foul smells and corrosion caused by the liberated hydrogen sulphide and destruction of emulsifiers, with consequent deterioration and instability of emulsions. Similar problems of industrial spoilage caused by obligate anaerobic sulphate-reducing bacteria are also encountered in paper-pulp sulphite processing liquids, water-based paints, oil-well injection water, process waters, electroplating solutions, in sealed water-circulating systems, for example heat-exchangers and cooling and heating water systems, hot water storage and delivery systems, and in water-seals of gasholders used for the storage of hydrocarbon gases.Spoilage caused by obligate anaerobic sulphate-reducing bacteria is a particularly troublesome problem in the water which collects at the bottom of fuel oil tanks and in oil-well injection water.

Oil-in-water emulsion metal-working fluids and aqueous solutions and emulsions of glycols used as metal-working fluids, e.g. machine-tool coolants and metal-rolling coolants, which comprise mineral or vegetable oils or glycols, emulsifying agents, corrosion inhibitors and water, with, if desired, fats, soaps and small quantities of special additives to serve as film plasticisers, anti-foaming agents and high pressure additives, are, as is well known, widely used for cooling and lubricating purposes in metal working operations. These fluids serve a number of functions in metal-working operations, including lubrication and reduction of temperature at the tool-work interface, promotion of tool life and removal of chips and swarf from the metal-working area and cooling sheet metal during rolling and pressing operations.For reasons of economy, it is essential that the metal-working fluid is retained in a recirculating system, so that the fluid may be re-used repeatedly. Such metal-working fluids normally contain from about 1% to about 1 5% by volume of oil and/or glycols and operate at temperatures from just above ambient to 40~60 C. Thus, metal-machining coolants fluids operate at temperatures just above ambient and their oil and/or glycol content varies from about 1% in grinding coolants, where heat dissipation to preserve dimensional accuracy is of prime importance, to about 15% in severe machining operations, where lubrication is an essential requirement, while metal-rolling coolant fluids usually have an oil and/or glycol content of 2 to 5% by volume and operate at higher temperatures, which are often 40--60"C. These metal-working fluids are readily susceptible to industrial spoilage by the growth of microorganisms and their oxygen levels frequently become very low, particularly when the fluid is not in use. This encourages the growth of obligate anaerobic sulphate-reducing bacteria resulting in serious problems of industrial spoilage caused by the degradation of alkyl sulphate and alkyl aryl sulphonate emulsifiers, breakdown of the emulsions, liberation of free oil and corrosion and foul smells caused by the liberation of hydrogen sulphide.

Combatting industrial spoilage caused by the growth of obligate anaerobic sulphate-reducing bacteria is, accordingly, of major importance in the recirculation and re-use of oil-in-water emulsion metal-working fluids and aqueous solutions and emulsions of glycols used as metal-working fluids.

Attempts have been made to combat industrial spoilage caused by the growth of microorganisms by the incorporation of many biocides but the properties required of a satisfactory biocide are extremely demanding and relatively few have been commercially successful. Thus, a biocide to be used to combat industrial spoilage caused by the growth of microorganisms, apart from being effective in killing or inhibiting the growth of the microorganisms, should, desirably, be non-toxic and non-irritant in use, have satisfactory stability over an adequate length of time, at the appropriate temperature ranges and pH ranges, be preferentially soluble in water to give adequate concentration in the aqueous phase where microorganisms growth takes place and not preferentially soluble in the oil or organic phase, be compatible with the system in question, for example without affecting adversely emulsion characteristics and stability, inducing foaming or frothing or itself affect the pH of the system and, when used in fuel oils or in metal-working, undesirable ash on combustion. Various phenols, e.g. p-chloro-m cresol and phenol, heavy metal compounds, compounds which liberate formaldehyde, and other biocidally active compounds have been used commercially to combat industrial spoilage caused by the growth of microorganism with varying degrees of success and the safety in use of many of them has been questioned. There is, accordingly, a continuing need for new methods of combatting industrial spoilage caused by the growth of microorganisms, more especially against spoilage caused by the growth of obligate anaerobic sulphate-reducing bacteria.

It is known that 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (hereinafter referred to as 'metronidazole'), 1 ,2-dimethyl-5-nitroimidazole (hereinafter referred to as 'dimetridazole'), 1-(2 hydroxypropyl)-2-methyl-5-nitroimidazole (hereinafter referred to as 'secnidazole') and 1-methy -2- isopropyl-5-nitroimidazole (hereinafter referred to as 'ipronidazole') are active against protozoa, amoebae and certain anaerobic bacteria which may infect humans and animals.Obligate anaerobic sulphate-reducing bacteria are not a recognised cause of infections in humans and animals and it has not hitherto been suggested that the aforesaid compounds would be effective against obligate anaerobic sulphate-reducing bacteria in human or veterinary medicine nor that they would be effective in combatting industrial spoilage caused by the aforesaid bacteria. Metronidazole, dimetridazole, secnidazole and ipronidazole would not, therefore, suggest themselves to those seeking new ways of combatting industrial spoilage caused by such microorganisms, nor would it be apparent that the compounds possess the combination of other properties necessary for them to be used successfully to combat industrial spoilage.

As a result of research and experimentation, it has now surprisingly been found that the nitroimidazole compounds dimetridazole, metronidazole, secnidazole and ipronidazole may be used to combat the growth of obligate anaerobic sulphate-reducing bacteria in liquid organic materials containing water or aqueous liquids containing organic materials. It is to be understood that the term 'nitroimidazole compounds' as used in the present specification means dimetridazole, metronidazole, secnidazole and ipronidazole. Accordingly, there is provided, as a feature of the present invention, a method for combatting industrial spoilage of those liquid organic materials containing water or aqueous liquids containing organic materials which are subject to spoilage caused by the growth of obligate anaerobic sulphate-reducing bacteria, for example petroleum hydrocarbon and vegetable oils and liquids containing them used in industry, e.g. fuel oils, lubricating oils, mineral oil-based hydraulic fluids, oil-in-water emulsions, for example metal-working fluids, water-in-oil emulsions, for example water-inoil emulsion hydraulic fluids, aqueous solutions and emulsions of glycols used as metal-working fluids, paper-pulp sulphite processing liquids, water-based paints, oil-well injection water, industrial process waters, electroplating solutions, sealed water-circulating systems, hot water storage and delivery systems and water seals of gas holders used for the storage of hydrocarbon gases, which comprises the incorporation into the aqueous phase of liquid organic materials containing water or aqueous liquids containing organic materials of an amount of at least one nitroimidazole compound according to the present invention sufficient to prevent the growth of obligate anaerobic sulphate-reducing bacteria.

The concentrations of nitroimidazole compound(s) which may be, separately or in combination, used according to the present invention are generally between 100 ppm and 500 ppm, and more especially 100 ppm, of the total volume of liquid organic material containing water or the aqueous liquid containing organic materials which is to be protected against industrial spoilage caused by the growth of obligate anaerobic sulphate-reducing bacteria but lower or higher concentrations may be used according to the particular problem of industrial spoilage in question and may be readily determined by appropriate testing.

The nitroimidazole compounds are not preferentially soluble in the organic phase of liquid organic materials containing water and aqueous liquids containing organic materials and are sufficiently soluble in the aqueous phase thereof to give concentrations effective in preventing the growth of obligate anaerobic sulphate-reducing bacteria, are non-toxic and non-irritant in use, in contrast to most of the agents which have hitherto been used to combat industrial spoilage caused by the growth of microorganisms, have satisfactory stability over an adequate length of time at the appropriate temperature and pH ranges, do not affect adversely the emulsion characteristics and stability of oil-inwater and water-in-oil emulsions, do not induce foaming or frothing or themselves affect pH and do not produce undesirable ash on combustion.

The nitroimidazole compounds according to the present invention may be added themselves to liquid organic materials containing water or aqueous liquids containing organic materials which it is desired to protect against industrial spoilage caused by the growth of obligate anaerobic sulphatereducing bacteria but are preferably added in the form of solutions, suspensions or emulsions in suitable solvents, for example water, glycols, e.g. ethylene glycol, ethyl oxalate, dimethylformamide, dimethylacetamide, liquid hydrocarbons, e.g. kerosene, and mixtures thereof, which may, if necessary, include one or more surface-active agents.An illustrative example of a suitable formulation is a suspension prepared by mixing microfine dimetridazole (25% weight/volume), Texofor M6 (4% weight/volume), Arylan CA (6% weight/volume), Attagel 50 (1.0% weight/volume), and kerosene (odourless) to 100% by volume.

Texofor M6 is an ethoxylated oleic acid containing 6 moles of ethylene oxide per mol of oleic acid; Arylan CA is calcium dodecylbenzenesulphonate; Attagel 50 is a swelling atapulgite clay.

According to a preferred feature of the present invention, there are provided metal-working concentrates suitable, on appropriate dilution with water, for the preparation of metal-working fluids which comprise oils and/or glycols, emulsifiers and corrosion inhibitors conventional in concentrates used in the preparation, by dilution with water, of metal-working fluids and an amount of nitroimidazole compound(s) sufficient to give the desired concentration of nitroimidazole compound(s) in the metalworking fluid after dilution with water, and generally from about 0.05% to 5% weight/volume of nitroimidazole compound(s).

If desired, the nitroimidazole compounds according to the present invention may be used in association with known agents for combatting industrial spoilage caused by the growth of microorganisms, for example phenols, e.g. p-chloro-m-cresol and phenol, heavy metals and compounds which liberate formaldehyde, in the method according to the present invention for combatting industrial spoilage caused by the growth of obligate anaerobic sulphate-reducing bacteria in liquid organic materials containing water and aqueous liquids containing organic materials. The nitroimidazole compounds according to the present invention will also combat industrial spoilage caused by nitrite and nitrate-reducing bacteria in liquid organic materials containing water and aqueous liquids containing organic materials.

The valuable properties of the nitroimidazole compounds according to the present invention in combatting the growth of obligate anaerobic sulphate-reducing bacteria in liquid organic materials containing water and aqueous liquids containing organic materials which are subject to spoilage caused by the growth of obligate anaerobic sulphate-reducing bacteria are demonstrated in the following Experiments 1 to 4.

EXPERIMENTS 1 to 4~Activity against obligate anaerobic sulphate-reducing bacteria Inhibition of the growth of obligate anaerobic sulphate-reducing bacteria.

Test Procedure {I) for obligate anaerobic sulphate-reducing bacteria The test compounds were incorporated, by the addition of the appropriate amounts of aqueous solutions containing 5000 ppm of dimetridazole or metronidazole, at concentrations of 50, 100, 250 and 500 ppm in an oil-in-water emulsion known to be heavily contaminated with obligate anaerobic sulphate-reducing bacteria. Samples of the contaminated oil-in-water emulsion to which no test compound had been added were maintained as controls. Samples from the contaminated oil immediately after addition of the test compounds and after 3, 5 and 24 hours were taken.

Iron sulphite agar (Oxoid Ltd) was prepared and poured into tubes (10 ml). The tubes were sterilised by autoclaving at a pressure of 15 Ibs per square inch for 15 minutes and sealed with Astell caps, which ensure an air-tight sea and hence allow anaerobic growth. The tubes were placed in a water-bath at 5O0C, which maintained the agar in a liquid state. Loopfuls (1/50 ml) of the samples of bacterially contaminated oil-in-water emulsions were then used as inoculum and the tubes were incubated at 370C for 5 days.

The effect of the test compounds on obligate anaerobic sulphate-reducing bacteria present in the contaminated oil-in-water emulsion was scored as follows: Score +++++completely black - no inhibition ++++good growth, but not completely black +++black/grey patches of inhibition and growth ++grey with black specks +clear except for a few black specks 0 clear - complete inhibition The following results were obtained: :~ EXPERIMENT 1 (a) Metronidazole Contact time with metronidazole Concentration of metronidazole Immediately 3 hours 5 hours 24 hours o (Control) +++++ +++++ +++++ ++++++++++ 50 ppm +++++ 100 ppm +++++ +++++ ++ + 250 ppm ++++ +++ O 0 500 ppm +++ ++ + 0 (b) Dimetridazole Concentration Contact time with dimetridazole of dimetridazole Immediately 3 hours 5 hours 24 hours 0 (Control) +++++ +++++ +++++ +++++ 50 ppm +++++ ++++ ++++ 100 ppm ++++ +++ ++ + 250 ppm ++++ ++ + 0 500 ppm +++ + 0 0 EXPERIMENT 2 (a) Metronidazole Concentration Contact time with metronidazole of metronidazole Immediately 3 hours 5 hours 24 hours 0 (Control) ++++ ++++ 100 ppm ++++ +++ + + 200 ppm ++++ ++ + + 500 ppm +++ + + 0 (b) Dimetridazole Concentration Contact time with dimetridazole of dimetridazole Immediately 3 hours 5 hours 24 hours 0 (Control) ++++ ++++ ++++ ++++ 100 ppm ++++ +++ + 0 200 ppm +++ + 0 0 500 ppm +++ 0 0 0 EXPERIMENT 3 (a) Metronidazole Concentration Contact time with metronidazole of metronidazole Immediately 3 hours 5 hours 24 hours 25 ppm ++++ +++ +++ ++++++ 50 ppm ++++ +++ +++ +++ 100 ppm ++++ +++ + + (b) Dimetridazole Concentration Contact time with dimetridazole of dimetridazole Immediately 3 hours 5 hours 24 hours 15 ppm ++++ +++ ++ ++ 50 ppm ++++ +++ ++ ++ 100 ppm ++++ +++ + + EXPERIMENT 4 [Test Procedure (II)] Flasks containing 100 ml of aqueous solutions containing 50, 100, 250 or 500 ppm of test compound were treated with an oil-in-water emulsion (0.1 ml) known to be heavily contaminated with obligate anaerobic sulphate-reducing bacteria. A control flask containing no test compound was also prepared. Samples from the control flask were taken immediately after addition of the oil-in-water emulsion and from all the flasks after 3, 5, 24 and 48 hours.

Iron sulphite agar (Oxoid Ltd) was prepared and poured into tubes (10 ml). The tubes were sterilised by autoclaving at a pressure of 15 Ibs per square inch for 1 5 minutes and sealed with Astell caps, which ensure an air-tight seal and hence allow anaerobic growth. The tubes were placed in a water-bath at 500C, which maintained the agar in a liquid state. Loopfuls (1/50 ml) of the sample of bacterially contaminated oil-in-water emulsions were then used as inoculum and the tubes were incubated at 370C for 7 days. The effect of the test compounds on obligate anaerobic sulphate-reducing bacteria present in the contaminated oil-in-water emulsion was scored as hereinbefore described.The following results were obtained:~ (a) Metronidazole Contact time with metronidazole Concentration of metronidazole Immediately 3 hours 5 hours 24 hours 48 hours o (Control) +++++ +++++ +++++ ++++t+++++ 50 ppm - +++++ ++++ +++ ++ 100 ppm - +++++ +++ + 0 250 ppm - ++++ ++ O 0 500 ppm -- +++ + O 0 (b) Dimetridazole Contact time with dimetridazole Concentration of dimetridazole Immediately 3 hours 5 hours 24 hours 48 hours 0 (Control) +++++ +++++ +++++ +++++ +++++ 50 ppm - +++++ ++++ +++ ++ 100 ppm - +++ ++ + 0 250 ppm ~ ++ + O 0 500 ppm + 0 0 0 (c) Ipronidazole Contact time with Ipronidazole Concentration of Ipronidazole Immediately 3 hours 5 hours 24 hours 48 hours 0 (Control) +++++ ++++ +++++ +++++ 50 ppm - +++++ ++++ +++ ++ 100 ppm - ++++ +++ ++ + 250 ppm - +++ ++ + O 500 ppm ~ +++ + 0 0 (d) Secnidazole Contact time with secnidazole Concentration of secnidazole Immediately 3 hours 5 hours 24 hours 48 hours 0 (Control) +++++ +++++ +++++ +++++ 50 ppm - +++++ +++++ ++++ 100 ppm - +++++ ++++ +++ + 250 ppm - ++++ ++ O 0 500 ppm ~ +++ + 0 0 (e) Kathon MW886 Contact time with Kathon MW886 Concentration of Kathon MW886 Immediately 3 hours 5 hours 24 hours 48 hours 0 (Control) +++++ +++++ +++++ +++++ +++++ 50 ppm - +++++ ++++ +++ ++ 100 ppm - +++++ ++++ +++ + 250 ppm - ++++ +++ ++ 0 500 ppm - +++ ++ + 0 (f) Grotan TK2 Contact time with Grotan TK2 Concentration of Grotan TK2 Immediately 3 hours 5 hours 24 hours 48 hours 0 (Control) +++++ +++++ +++++ +++++ 250 ppm - +++++ ++++ +++ ++ 500 ppm - ++++ ++++ +++ ++ 750 ppm - +++ ++ + 0 1000 ppm - +++ + 0 0 (g) Bodoxin Contact time with Bodoxin Concentration of Bodoxin Immediately 3 hours 5 hours 24 hours 48 hours 0 (Control) +++++ +++++ +++++ +++++ 250 ppm +f+++ +++++ ++++ +++ ++ 500 ppm - ++++ +++ ++ + 750 ppm +++ ++ + 0 1000 ppm - ++ + 0 0 Kathon MW886, Grotan TK2 and Bodoxin are commercially available industrial biocides used to control contamination by obligate anaerobic sulphate-reducing bacteria.

The results obtained in Experiments 1,2,3 and 4 confirm that metronidazole, dimetridazole, ipronidazole and secnidazole inhibit sulphide production by obligate anaerobic sulphate-reducing bacteria, the effective concentration in these Experiments 1 to 4 being about 100 ppm. The effective concentration of Kathon MW886, which can cause severe skin and eye irritation, is also about 100 ppm. For Grotan TK2 and Bodoxin, the effective concentration is higher, at about 500 ppm.

The compatibility of nitroimidazoles according to the present invention with oil-in-water emulsion metal-working fluids prepared from commercially available metal-working concentrates are demonstrated in the following Experiments 5 to Sin which the commercially available metal-working concentrates are:~ Concentrate A: a general purpose cutting oil emulsion - used at 2 to 3% v/v (volume/volume) concentration; Concentrate B: a general purpose cutting oil - used at 1 to 4% v/v concentration; Concentrate C: an aluminium rolling oil which becomes unstable at the 'bite' of the rollers and reforms afterwards - used at 5% v/v concentration; Concentrate D: a semi-synthetic cutting oil formulation - used as a cutting oil at 1 to 2% v/v concentration; Concentrate E: a general purpose cutting oil - used at 3 to 5% v/v concentration; Concentrate F: a cutting oil used for high grade machine finishing - used at 3% v/v concentration; Concentrate G: a non-oil based water-containing synthetic grinding fluid - used at 2% v/v concentration.

EXPERIMENT 5 Emulsion stability Cutting and rolling oils used in the form of oil-in-water emulsions must retain emulsion-stability in use. This experiment is designed to assess the stability of such oil-in-water emulsions by recording the tendency of oil droplets to coalesce, which is a feature of instability.

Oil-in-water emulsions containing commercially available oil concentrates were prepared by the following procedure [Institute of Petroleum Standard Test (IP) 263/70 ] :- Distilled water and the oil concentrate were separately brought to a temperatureof 20+40C before mixing. The appropriate volume of water was stirred in a 500 ml conical flask, using a magnetic stirrer, at a speed sufficient to create a vortex just deep enough to reach the bottom of the flask.The appropriate volume of oil concentrate and the appropriate volumes of a solution containing 5000 ppm of mentronidazole, dimetridazole, or ipronidazole in water to give 50, 100, 250 and 500 ppm of metronidazole, dimetridazole or ipronidazole in the oil-in-water emulsion (total volume 200 ml) were then added quickly from a hypodermic syringe (without a needle) and stirring was continued for 2 minutes after the additions were complete. Oil-in-water emulsions containing no nitroimidazole compound were similarly prepared as controls. The oil emulsion was then allowed to stand at ambient temperature for 24 hours.

A sample of the oil emulsion thus obtained was diluted to 1:2000 with Steriflex No 1 Saline (Allen and Hanbury's Ltd) and the 'oversize' oil droplet size distribution in an 0.5 ml sample of this dilution was determined using a Coulter Counter (100 m orifice) (Coulter Electronics Ltd) connected to a P64 Analyser and Plotter. The oil-in-water emulsions were then left for 7 days and the oil droplet size distribution was again determined using the same setting of the Coulter Counter, so that direct comparisons could be made. As is normal with oil-in-water emulsions which have been left undisturbed for several days, separation into oil, curds and cream had occurred between the two determinations and prior to sampling of the second determination, the flasks were shaken for 30 seconds, rotating alternatively clockwise and anti-clockwise.

The following results were obtained:~ (a) Oil-in-water emulsion containing 2% v/v of Concentrate A Metronidazole: an increase in 2~5,um3 droplets at 100 ppm of metronidazole after 7 days indicated very slight emulsion in stability Dimetridazole: emulsion stability after 7 days had improved slightly, particularly at 50 ppm of dimetridazole Ipronidazole: no significant decrease in emulsion stability was observed after 7 days.

(b) Oil-in-water emulsion containing 1% vlv Concentrate B Metronidazole: emulsion stability after 7 days had improved slightly.

Dimetridazole: slight instability after 7 days in the oil-in-water emulsion containing 250 ppm of dimetridazole was detected.

Ipronidazole: no significant change in emulsion stability was observed after 7 days.

(c) Oil-in-water emulsion containing 3% v/v of Concentrate F Metronidazole: no change in emulsion stability after 7 days was detected.

Dimetridazole: no change in emulsion stability after 7 days was detected.

(d) Oil-in-water emulsion containing 3% v/v of Concentrate E Metronidazole: a slight increase in emulsion stability after 7 days was detected.

Dimetridazole: emulsion stability after 7 days was within satisfactory limits.

Ipronidazole: a very slight decrease in emulsion stability was observed after 7 days.

(e) Oil-in-water emulsion containing 1% v/v of Concentrate D Metronidazole: no change in emulsion stability after 7 days was detected.

Dimetridazole: a slight increase in emulsion stability after 7 days was detected.

Ipronidazole: a very slight decrease in stability after 7 days was observed in the oil-in-water emulsion containing 250 ppm of ipronidazole.

(f) Oil-in-water emulsion containing 2% v/v of Concentrate G Ipronidazole: no significant change in emulsion stability was detected after 7 days.

(g) Oil-in-water emulsion containing 2% v/v of Concentrate C Metronidazole: no change in emulsion stability was detected.

Dimetridazole: no change in emulsion stability was detected.

Ipronidazole: a very slight decrease in emulsion stability was detected after 7 days.

The results show that the incorporation of metronidazole, dimetridazole and ipronidazole at concentrations of 50, 1 00, 250 and 500 ppm in the oil-in-water emulsion had no significant adverse effect on emulsion stability.

EXPERIMENT 6-Corrosiveness This experiment is designed (IP 287/74) to assess the ability of oil-in-water emulsions to prevent rusting of machines and compounds during manufacturing operations, by the following procedure:~ Cast iron chips (Herbert Machine Tools Ltd. P.O. Box 30, Edgwick Works, Coventry, England) were washed in acetone (Reagent Grade) and dried in a warm cabinet at 480C. During washing, the chips were not touched by hand. When dry, the chips were sieved on a 25 mesh sieve, material passing through the sieve being rejected. An outline 35 mm square was drawn in pencil in the centre of a filter paper (Whatman No 5, Qualitative, 90 mm), which was then placed in the bottom of a petri dish. With the aid of a spatula and former, the marked 35 mm square area was then covered with a single layer of chips.The former was then removed and 2 ml quantities of oil-in-water emulsions (prepared by the procedure described in Experiment 5) containing a range of proportions of oil concentrates were pipetted onto the chips, so that the entire area of the petri dish was covered and the chips were thoroughly wetted. A cover was then placed over the petri dish. After 2 hours, the filter paper was removed from the dish, washed with tap water and left to dry. A transparent grid was then placed over the outline 35 mm square test area on the dry filter paper and the area of staining was assessed and expressed as a percentage. In order to facilitate assessment, the percentage area stained should be greater than 10% and less than 60%. The dilution of each oil concentrate which there was a significant increase in stained area (known as the 'breakpoint') was determined.For each oil concentrate, a series of oil-in-water emulsions containing the breakpoint proportion of oil concentrate and 50, 100, 250 and 500 ppm of metronidazole, dimetridazole or ipronidazole were then prepared and each oil-in-water emulsion was tested by the procedure described above.

The following results were obtained:~ (a) Oil-in-water emulsion containing ? 1/2% v/v Concentrate A

Area stained expressed as a percentage Concentration of Nitroimidazole (ppm) Metronidazole Dimetridazole 0 (Control) 8 2 50 10 4 100 10 6 250 1 8 500 0 10 A slight decrease in the area stained was observed at 250 ppm and 500 ppm of metronidazole. Repeat experiments confirmed this observation. A slight increase in the area stained was observed with dimetridazole.

(b) Oil-in-water emulsion containing 4% w/v of Concentrate B

Area stained expressed as a percentage Concentration of Nitroimidazole (ppm) Metronidazole | Dimetridazole Ipronidazole 0 (Control) 15 i 15 | so 100 30 25 60 250 35 | 30 40 500 40 40 50 A slight increase in the area stained was observed with both metronidazole and dimetridazole. A slight decrease in the area stained was observed with ipronidazole.

(c) Oil-in-water emulsion containing 5% v/v of Concentrate C

Area stained expressed as a percentage Concentration of Nitroimidazole (ppm)| Metronidazole | Dimetridazole | 0 (Control) 50 20 50 60 20 | 100 50 20 | 250 70 40 500 70 50 A slight increase in the area stained was observed with both metronidazole and dimetridazole.

(d) Oil-in-water emulsion containing 2% v/v Concentrate D

Area stained expressed as a percentage Concentration of Nitroimidazole (ppm) Metronidazole Dimatridazole Ipronidazole 0 (Control) 40 35 30 50 40 40 25 100 40 50 ! 55 250 40 50 30 500 40 40 15 No change in rust staining was detected with metronidazole; a slight increase in staining was detected with dimetridazole; with ipronidazole, a slight increase in staining was found at 100 ppm and a slight decrease at 50. 250 and 500 ppm.

(e) Oil-in-water emulsion containing 3% v/v of Concentrate F

Area stained expressed as a percentage Concentration of Nitroimidazole (ppm) Metronidazole Dimetridazole 0 (Control) 40 45 50 60 20 100 30 45 250 30 30 500 30 25 A slight variation in staining was observed with both metronidazole and dimetridazole.

(f) Oil-in-water emulsion containing 5% v/v of Concentrate E

| Area stained expressed as a percentage Concentrate of Nitroimidazole (ppm) Metronidazole Dimetridazole 0 (Control) 25 20 50 30 25 100 25 30 250 20 35 500 20 45 These results indicated no significant interference by metronidazole and dimetridazole at the concentrations tested with the anti-corrosive properties of the oil-in-water emulsions tested.

(g) Oil-in-water emulsion containing 2% v/v of Concentrate G

Area stained expressed as a percentage Concentrate of Nitroimidazole (ppm) Ipronidazole 0 (Control) 15 50 3 100 3 250 3 500 3 A slight decrease in the area stained was observed.

EXPERIMENT 7-- effect on pH Oil-in-water emulsions were prepared as described in Experiment 5 and their pH values were measured at 1 and 7 days after preparation, using a pH meter fitted with an Ingold electrode.

The following results were obtained (a) Oil-in-water emulsion containing 2 1/2% v/v of Concentrate A

pH Metronidazole Dimetridazole Ipronidazole Concentration of Nitroimidazole (ppm) 1 day 7 days 1 day 7 days 1 day 7 days 0 (Control) 8.8 8.6 8.9 8.8 9.76 9.21 50 8.8 8.7 8.9 8.8 | 9.97 9.83 100 9.0 8.6 8.9 8.8 9.76 9.30 250 8.9 8.6 8.8 8.8 9.88 9.50 500 8.8 8.6 8.8 8.8 9.95 9.81 (b) Oil-in-water emulsion containing 4% v/v of Concentration B

pH Metronidazole Dimetridazole Ipronidazole Concentration of Nitroimidazole (ppm) 1 day 7 days 1 day 7 days 1 day 7 days 0 (Control) | 9.4 9.0 9.2 9.0 9.78 8.89 50 9.2 9.0 9.2 9.0 9.81 9.17 100 9.2 8.9 9.2 9.0 ; 9.89 9.20 250 9.2 9.0 9.2 9.0 10.10 9.35 500 | 9.2 9.0 | 9.2 9.0 9.94 9.26 (c) Oil-in-water emulsion containing 5% v/v of Concentrate C

pH Metronidazole Dimetridazole Ipronidazole Concentration of Nitroimidazole (ppm) 1 day 7 days 1 day 7 days 1 day 7 days 0 (Control) 8.2 8.0 8.2 8.0 8.40 8.46 50 8.2 8.0 8.2 8.1 8.43 8.53 100 8.1 8.0 8.2 8.0 8.41 8.50 250 L 8.2 8.1 8.2 8.0 8.43 8.50 500 8.2 8.0 8.2 8.0 8.68 8.81 (d) Oil-in-water emulsion containing 2% v/v of Concentrate D

pH Metronidazole Dimetridazole Ipronidazole Concentration of Nitroimidazole (ppm) 1 day 7 days 1 day 7 days 1 day 7 days 0 (Control) 9.4 9.2 9.4 9.2 9.63 9.00 50 9.4 9.2 9.3 9.2 9.72 9.10 100 9.4 9.0 9.4 9.2 9.69 9.18 250 9.4 9.2 9.4 9.2 9.85 9.26 500 9.4 9.2 9.4 9.2 9.81 9.14 (e) Oil-in-water emulsion containing 5% v/v of Concentrate E

pH Metronidazole Dimetridazole ipronidazole Concentration of Nitroimidazole (ppm) 1 day 7 days 1 day 7 days 1 day 7 days 0 (Control) 9.5 9.2 9.4 9.2 9.94 9.53 50 9.4 9.2 9.4 9.3 10.01 9.60 250 9.4 9.2 9.5 9.3 9.98 9.54 500 9.5 9.3 9.5 9.3 10.02 9.60 (f) Oil-in-water emulsion containing 3 1/2% v/v of Concentrate F

pH Metronidazole Dimetridazole Concentration of Nitroimidazole (ppm) 1 day 7 days 1 day 7 days 0 (Control) 9.1 9.0 9.0 8.9 50 9.0 8.9 9.1 8.9 100 9.1 8.9 9.1 9.0 250 9.1 9.0 9.0 8.9 500 9.1 9.0 9.0 9.0 (g) Oil-in-water emulsion containing 2% v/v of Concentrate G

pH Ipronidazole Concentration Nitroimidazole (ppm) 1 day 7 days 0 (Control) 10.01 9.06 50 10.18 9.54 100 10.12 9.48 250 10.12 9.56 500 10.16 9.56 These results indicate that metronidazole, dimetddazole and ipronidazole at the concentrations tested do not significantly affect the pH of the oil-in-water emulsions tested.

EXPERIMENT 8~frothing and foaming characteristics This experiment is designed (IP 312/74) to assess the frothing and foaming characteristics of oilin-water emulsions by the following procedure:~ Oil-in-water emulsions of oil concentrates were prepared by the procedure described in Experiment 5, but replacing the distilled water by Synthetic Hard Water (a calcium sulphate solution of total hardness equivalent to 200 + 10 ppm in terms of calcium carbonate, prepared by dissolving 0.344 g of calcium sulphate (Ca SO4.2H20) per litre of distilled water). A 100 ml portion of oil-in-water emulsion was placed in a 250 ml capacity measuring cylinder (graduated and conforming to BS 604:1 952), stoppered and shaken vigorously for 15 seconds.After shaking, the cylinder was placed in a vertical position and the volume of foam and liquid was recorded immediately and at 1, 5, 10 and 1 5 minutes after shaking (Froth may also form above the foam but this froth tends to break down, leaving air pockets and the height of the froth in the cylinder is difficult to decide. The foam, being more dense, can be seen readily, and for convenience, the foam level was recorded).

Concentrate C formed a precipitate when emulsified using Synthetic Hard Water. This precipitate did not alter with the incorporation of metronidazole, dimetridazole or ipronidazole. The test was repeated using distilled water instead of Synthetic Hard Water and it is the results of this dilution of Concentrate C with distilled water which are given below).

The following results, in which 'Volume' indicates the volume of foam and liquid were obtained:~ (a) Oil-in-water emulsion containing 1% v/v of Concentrate B

Volume - on standing (minutes) Concentration of metronidazole (ppm) O 1 1 5 | 10 15 0 (Control) 120 100 100 | 100 100 50 120 101 100 100 100 100 120 100 100 100 100 250 120 100 100 100 100 500 125 101 100 100 100 Volume -- on standing (minutes) Concentration of dimetridazole (ppm) 0 1 5 10 15 0 50 120 100 100 100 100 100 120 100 100 100 100 250 130 100 100 100 100 500 130 100 100 100 100 Volume - on standing (minutes) Concentration of ipronidazole (ppm) O 1 5 10 15 15 (Control) 202 174 152 142 131 50 182 176 164 132 130 100 194 182 154 138 134 250 198 188 158 134 132 500 198 184 154 141 131 (b) Oil-in-water emulsion containing 1% v/v of Concentrate D

Volume - on standing (minutes) Concentration of metronidazole (ppm) O 1 5 10 15 0 (Control) +250 +250 250 150 140 50 +250 +250 250 140 125 100 +250 +250 1 235 150 135 250 +250 +250 250 140 130 500 +250 +250 250 145 130 Volume - on standing (minutes) Concentration of dimetridazole (ppm) O 1 5 10 15 0 (Control) +250 +250 250 140 I 125 50 +250 +250 250 150 130 100 +250 +250 240 135 120 250 +250 +250 250 150 135 500 +250 +250 250 145 125 Volume -- on standing (minutes) Concentration of Ipronidazole (ppm) 0 1 5 10 15 0 (Control 190 190 188 146 132 50 193 188 146 138 130 100 206 204 126 124 122 250 192 176 162 140 130 500 212 190 140 128 120 ('+250' = foam level above the 250 ml. level) (c) Oil-in-water emulsion containing 3% v/v of Concentrate F

Volume - on standing (minutes) Concentration of metronidazole (ppm) O 1 5 10 15 0 (Control) 150 108 105 104 104 50 145 110 108 106 106 100 130 112 108 106 104 250 140 108 106 104 104 500 160 108 104 104 102 Volume -- on standing (minutes) | Concentration of dimetridazole(ppm) 0 1 5 10 15 0 (Control) 145 106 104 104 102 50 140 108 106 104 104 100 150 108 108 106 104 250 145 110 106 104 104 500 160 108 104 104 102 (d) Oil-in-water emulsion containing 3% v/v of Concentrate E

Volume -- on standing (minutes) Concentration of metronidazole (ppm) 0 1 5 10 15 0 (Control) +250 +250 220 170 150 50 +250 +250 230 170 150 100 +250 +250 230 150 140 250 +250 +250 +250 160 140 500 +250 +250 240 180 | 140 Volume -- on standing (minutes) Concentration of dimetridazole (ppm) O 1 5 10 15 0 (Control +250 +250 210 140 140 | 50 +250 +250 220 170 150 100 +250 +250 210 180 160 250 +250 +250 240 180 160 500 +250 +250 220 170 150 Volume - on standing (minutes) Concentration of ~015 Ipronidazole (ppm) 0 | 1 5 10 15 | O (Control) 168 108 102 102 100 50 172 122 100 168 118 108 104 102 -- 250 166 120 108 106 102 500 | 192 114 106 104 102 (e) Oil-in-water emulsion containing 2% v/v of Concentrate A

Volume - on standing (minutes) Concentration of metronidazole (ppm) 0 1 5 10 15 0 (Control) 140 103 101 100 100 50 130 106 | 104 101 100 100 140 104 102 100 100 250 135 106 104 101 1 100 500 140 106 | 105 104 | 102 Volume - on standing (minutes) Concentration of dimetridazole (ppm) O 1 5 10 15 0 (Control) 135 103 101 101 | 101 50 | 150 110 101 101 100 100 155 112 102 100 100 250 130 | 101 101 100 100 500 140 104 101 100 100 Volume - on standing (minutes) Concentration of Ipronidazole (ppm) 0 1 5 10 15 0 (Control) 246 122 114 110 106 50 230 116 114 110 102 100 212 140 128 118 114 250 202 128 112 110 106 500 +250 116 108 104 100 (f) Oil-in-water emulsion containing 3% v/v of Concentrate C

Volume - on standing (minutes) Concentration of metronidazole (ppm) O 1 5 10 15 0 (Control) 101 100 100 100 100 50 100 100 100 100 100 100 100 100 100 100 100 250 100 100 100 100 100 500 100 I 100 1 100 100 100 Volume -- on standing (minutes) Concentration of dimetridazole (ppm) 0 1 5 10 15 0 | (Control) 102 100 100 100 100 50 100 100 100 100 100 100 100 100 100 100 100 250 100 100 100 100 100 500 100 100 Volume -- on standing (minutes) Concentration of ipronidazole (ppm) O 1 5 10 15 (Control) 152 132 116 110 | 108 50 142 112 108 106 104 100 136 110 106 104 102 250 142 112 108 104 102 500 152 114 110 108 104 (g) Oil-in-water emulsion containing 2% v/v of Concentrate G

Volume - on standing (minutes) Concentration of Ipronidazole (ppm) O 1 5 10 15 0 (Control 100 100 100 100 100 50 100 100 100 100 100 100 '' 100 100 100 100 100 250 100 100 100 100 100 500 100 100 100 100 100 These results show that the incorporation of metronidazole, dimetridazole or ipronidazole at the indicated concentrations made no significant difference to the frothing and foaming characteristics of the oil-in-water emulsions tested.

The following Example illustrates metal-working concentrates suitable, on appropriate dilution with water, for the preparation of metal-working fluids according to the present invention.~ EXAMPLE A suspension is prepared by mixing microfine dimetridazole (25% weight/volume), Texofor M6 (4% weight/volume), Arylan CA (6% weight/volume), Attagel 50 (1.0% weight/volume) and kerosene (odourless) to 100% by volume. One litre of this concentrate is mixed with 99 litres of a general purpose cutting oil concentrate to give 100 litres of a cutting oil concentrate containing 2500 ppm of dimetridazole. This concentrate (100 litres) may be diluted with 2400 litres of water to give 2500 litres of metal-working fluid containing 100 ppm of dimetridazole.

Claims

1. A method for combatting industrial spoilage of liquid orgaic materials containing water or aqueous liquids containing organic materials caused by obligate anaerobic sulphate-reducing bacteria which comprises the incorporation into the aqueous phase of liquid organic materials containing water or aqueous liquids containing organic materials of an amount of one or more nitroimidazole compound(s) selected from dimetridazole, metronidazole, secnidazole and ipronidazole in an amount sufficient to prevent the growth of obligate anaerobic sulphate-reducing bacteria.

2. A method according to claim 1 wherein the liquid organic materials containing water or aqueous liquids containing organic materials are petroleum hydrocarbon or vegetable oils containing water, or aqueous liquids containing petroleum hydrocarbon or vegetable oils, used in industry.

3. A method according to claim 1 or 2 wherein the liquid organic materials containing water or aqueous liquids containing organic materials are fuel oils, lubricating oils, mineral oil-based hydraulic fluids, oil-in-water emulsions, water-in-oil emulsions, aqueous solutions and emulsions of glycols used as metal-working fluids, paper-pulp sulphite processing liquids, water-based paints, oil-well injection water, industrial process waters, electroplating solutions, sealed water-circulating systems, hot water storage and delivery systems and water seals of gasholders used for the storage of hydrocarbon gases.

4. A method according to claim 3 wherein the oil-in-water emulsions are metal-working fluids.

5. A method according to claim 3 wherein the water-in-oil emulsions are water-in-oil emulsion hydraulic fluids.

6. A method according to any one of claims 1 to 5 wherein a solution, suspension or emulsion comprising one or more nitroimidazole compound(s) selected from dimetridazole, metronidazole, secnidazole and ipronidazole and water, glycols, ethyl oxalate, dimethylformamide, dimethylacetamide or liquid hydrocarbons or mixtures thereof and, if necessary, one or more surface active agents, is added to liquid organic materials containing water or aqueous liquids containing organic materials.

7. A method according to any one of claims 1 to 6 wherein the amount of one or more nitroimidazole compound(s) selected from dimetridazole, metronidazole, secnidazole and ipronidazole incorporated into the aqueous phase of liquid organic materials containing water or aqueous liquids containing organic materials is from 100 ppm to 500 ppm of the total volume of the liquid organic materials containing water or the aqueous liquids containing organic materials.

8. A method according to any one of claims 1 to 6 wherein the amount of one or more nitroimidazole compound(s) selected from dimetridazole, metronidazole, secnidazole and ipronidazole incorporated into the aqueous phase of liquid organic materials containing water or aqueous liquids containing organic materials is 100 ppm of the total volume of the liquid organic materials containing water or the aqueous liquids containing organic materials.

9. A method according to any one of claims 1 to 8 wherein one or more nitroimidazole compound(s) selected from dimetridazole, metronidazole, secnidazole and ipronidazole are used in association with one or more known agents for combatting industrial spoilage of liquid organic materials containing water or aqueous liquids containing organic materials.

10. A method according to claim 9 wherein the known agents are one or more of p-chloro-mcresol, phenol, heavy metals and compounds which liberate formaldehyde.

11. A method according to any of claims 1 to 10 wherein the obligate anaerobic sulphatereducing bacteria are one or more of Desulfovibrio desulfuricans, Desulfovibrio gigas and Desulfotomaculum nigrificans.

12. A method according to any one of claims 1 to 11 wherein the nitroimidazole compound is dimetridazole.

13. A method according to any one of claims 1 to 11 wherein the nitroimidazole compound is metronidazole.

14. A method according to claim 1 for combatting industrial spoilage of liquid organic materials containing water or aqueous liquids containing organic materials caused by obligate anaerobic sulphate-reducing bacteria substantially as herein described.

15. Metal-working concentrates suitable, on appropriate dilution with water, for the preparation of metal-working fluids which comprise oils and/or glycols, emulsifers and corrosion inhibitors conventional in concentrates used in the preparation, by dilution with water, of metal-working fluids and an amount of one or more nitroimidazole compound(s) selected from dimetridazole, metronidazole, secnidazole and ipronidazole sufficient to give a concentration of nitroimidazole compound(s), in the metal-working fluid after dilution with water, sufficient to prevent the growth of obligate anaerobic sulphate-reducing bacteria.

16. Metal-working concentrates according to claim 15 wherein the amount of nitroimidazole compound(s) incorporated in the concentrates is from 0.05% to 5% weight/volume.

17. Metal-working concentrates according to claim 15 or 16 wherein the nitroimidazole compound is dimetridazole.

18. Metal-working concentrates according to claim 15 or 16 wherein the nitroimidazole compound is metronidazole.

19. Metal-working concentrates according to claim 15 containing one or more nitroimidazole compound(s) selected from dimetridazole, metronidazole, secnidazole and ipronidazole substantially as hereinbefore described with special reference to the foregoing Example.