NZ587851A - Method Of Preparing A Stabilised Chlorine Dioxide Solution - Google Patents

Abstract

Disclosed is a method of preparing a stabilised chlorine dioxide solution, comprising the steps: (i) Take 500 grams of 80 percent sodium chlorite and dissolve in water (ii) add water to a two hundred litre container and (iii) add the solution prepared at step (i); (iv) at a level of one hundred Iitres of water add 500 - 1000 mililitres of HCI at a concentration of 32 percent weight per volume (v) at a water level of one hundred and fifty Iitres add a further 25grams of sodium chlorite and add one litre 16 percent weight per volume solution of HCI to the water and (vi) fill to 200 Litres. Further disclosed is a method for the control or suppression of infection in a non-human which comprises applying to a desired location the stabilised chlorine dioxide solution prepared by said method, and the use of stabilised chlorine prepared by said method in the manufacture of a biocidal composition for the control or suppression of infection.

Description

Patents Form # 5 NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION AFTER PROVISIONAL NO.: 587851 PRIORITY DATE : 8 September 2010 TITLE : Stabilised Chlorine Dioxide Solution We, SOUTHWELL IP LIMITED Address: 550 Tremaine Avenue, Palmerston North, New Zealand, 4410 Nationality: A body corporate organized and existing under the laws of New Zealand do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: 401766NZA_Cap_20110902_1105_EMM.doc FEE CODE 1050 Field of the Invention The invention relates to a stabilized solution of chlorine dioxide and the numerous uses of the solution in many areas of industry.

Background Sanitizers are well known today and in frequent use. Chlorine dioxide, for example, is a well known disinfectant sanitizer and water treatment product. A major problem with the uses of 1 5 carbon dioxide however is its delivery system. Until recently the only way of manufacturing chloride dioxide was by means of a generator. Two containers, one containing an acid the other a salt, were mixed together in a chamber and chlorine dioxide gas was generated and then metered into the water supply. For field applications this is not a satisfactory state of affairs.

The discovery of chlorine dioxjde is generally credited to Sir Humphrey Davy, who reported the results of the reaction of potassium chlorate with sulfuric acid in the early 1800's. Chlorine dioxide today is generated for smaller applications by the reaction of sodium chlorite with chlorine, via either gaseous chlorination (Equation 1) or the reaction of sodium hypochlorite with hydrochloric acid (Equation 2).

Cl2 + 2NaCI02 -> 2CI02 + 2NaCI (1) HCI + NaOCI + 2NaCI02 -> 2CI02 + 2NaCI + NaOH (2) This chemistry was due to the pioneering efforts of J. F. Synan, J. D. MacMahon, and J. P. Vincent, of Mathieson Chemical Company, now Olin Corporation. In 1944, the generation of chlorine dioxide to control taste and odor problems at a potable water facility at Niagara Falls, 30 N.Y., was reported.

This first successful application led to its use in other municipal potable water treatment facilities which had similar problems. Over the next 25 years researchers compared the disinfection efficiency of chlorine dioxide to that of the industry standard, chlorine.

In the mid to late 70's, researchers linked chlorination of potable water to increased cancer 35 mortality rates. This increase in cancer mortality was tied to the production of trihalomethanes, THM's. The USEPA established 0.1 ppm as the maximum THM containment level for drinking water. Research in the area of THM reduction in potable water led to the EPA in 1983 suggesting the use of chlorine dioxide as an effective means of controlling THM's.

In 1986, there was an estimated 200 - 300 chlorine dioxide applications for potable water 40 treatment in the USA, and applications in Europe numbered in the thousands. 2 Chlorine dioxide is being used increasingly to control microbiological growth in a number of different industries, including the dairy industry, the beverage industry, the pulp and paper industries, the fruit and vegetable processing industries, various canning plants, the poultry industry, the beef processing industry, and miscellaneous food processing applications. It is seeing increased use in municipal potable water treatment facilities and in industrial waste 10 treatment facilities, because of its selectivity towards specific environmentally-objectionable waste materials, including phenols, sulfides, cyanides, thiosulfates, and mercaptans. It is being used in the oil and gas industry for down-hole applications as a well stimulation enhancement additive. Today, domestic industrial applications number in the thousands.

With the recent trend towards elimination of gaseous chlorine from the industrial plant site, there 15 are increasing interests in exploring all the various alternatives to gaseous chlorine.

Acidified Sodium Chlorite, Stabilised Chlorine Dioxide and Chlorine dioxide in aqueous diluent, differences Acidified Sodium Chlorite (ASC) Is a weak colourless liquid with a, mild, chlorine like odour that is produced by adding a weak acid to solution of sodium chlorite (NaCI02). The active ingredient (at pH 2.3 to 3.2) consists mainly of chlorous acid (HCI02) in equilibrium with Chlorite ion (CI02") and H+, ASC in solution consists mainly of chlorite ions (65 to 95% at pH 2.3 to 3.2, respectively, H+ ions and chlorous 25 acid (35 to 45%) at pH 2.3 to 3.2, respectively. At pH>7 chlorine dioxide is the primary species present slowly decomposes to chlorate and chloride.

Chlorine dioxide is a relatively soluble compound with any that is generated in a fresh solution of ASC (generally) <3 ppm) tending to remain in solution. If the ASC solution is being sprayed, 30 any chlorine dioxide in the solution is usually immediately off-gassed, with greater off gassing as spray particle size decreases (i.e. the surface area to volume ratio increases).

The use of ASC (depending on pH) may result in the production of the following four primary chlorine compounds and chloride (CI") when a food grade acid is mixed with sodium chlorite.

Chlorite (CI02") chlorate (CI03), chlorous acid (HCI02) and chlorine dioxide (Cl02) Acidified Sodium Chlorite Chemistry ASC chemistry is the chemistry of chlorous acid (HCIO2) 40 Oxidation States of Chlorine CI04" +7 Perchlorate ion CIO3" +5 Chlorate Ions 3 CI02 +4 Chlorine Dioxide Chlorite ions Hypochlorite ion Chlorine (molecular) Chlorite ion CI02" CIO or OCI +3 +1 CI2 0 CI" -1 Stabilised Chlorine Dioxide Stabilised chlorine dioxide is a misleading term that is unfortunately in widespread use. There are only trace amounts of chlorine dioxide in "stabilised chlorine dioxide". The correct description of this is, "stabilised chlorite". The chlorite is stabilised with a buffer and peroxide at a pH of about 7. Though chlorite, or stabilised chlorite is also an oxidising agent, it is not nearly as powerful as chlorine dioxide. Chlorine Dioxide, unlike chlorite, is a gas, the term "active" chlorine dioxide is used to distinguish between the real and unreal.

There is also a great deal of confusion relating to so-called "stabilized chlorine dioxide" solutions, which have little or none of the free CI02 molecule, but which predominate instead in chlorite ion. The claim is made that during use, the unstable chlorite can lead to a slow generation of CI02 but not with sufficient rapidity to provide any significant CI02 activity. The "stabilisation" of chlorine dioxide, by reaction of the CI02 with peroxides to form chlorite, has been taught in a number of patents, including those of Wentworth (U.S. Pat. No. 3,123,521) and McNicholas (U.S. Pat. No. 3,271,242). Other attempts to stably contain CI02 are found in U.S. Pat. No. 4,829,129, in which the molecule is claimed to be complexed with an organic polymer, and in U.S. Pat. No. 4,861,514, where CI02is apparently maintained in a steady-state concentration, after its slow formation over many days, in a thickened aqueous solution comprising a gelling agent, a chlorite salt, and an aldehyde or acetal. In neither of these two patents does the resulting composition provide a simple stable solution, of freely-available CI02i, appropriate for easy disinfecting or deodorising applications, without the presence of other solutes necessary for CI02 stabilisation. In addition, the application of the referenced compositions to a substrate intended for disinfection, would leave significant levels of dried residue upon evaporation of the aqueous solvent.

Active Chlorine Dioxide The preferred method of manufacturing CIO2, because it guarantees the best conversion to Chlorine Dioxide, and, limits, as much as possible the formation of by-products, is: 5NaCI02 + 4 HCI -+ 4 CIO2 + 5 NaCI + 2 H20.

Some very harmful substances—dioxins and furans, for example, and also trihalomethanes can be formed when chlorine products come in contact with organic matter, such as leaves and dirt. Dioxins and furans, both reasonably anticipated to be human carcinogens by the International Agency for Research on Cancer (IARC), are organochlorine compounds similar in structure to 4 PCBs. They biodegrade very slowly and therefore build up in the bodies of animals and humans; dioxin and furan have even been detected in breast milk samples. Trihalomethanes, including the carcinogen chloroform are formed when chlorine reacts with carbon-containing organic matter. They can increase the risk of cancer and may damage the liver, kidneys, and nervous system, and increase rates of miscarriage and birth defects.

Sodium hypochlorite and chlorine production Sodium hypochlorite is another well known sanitizer and may be prepared by absorbing chlorine gas in cold sodium hydroxide solution: 2NaOH + Cl2 -»• NaCI + NaOCI + H20 Sodium hydroxide and chlorine are commercially produced by the chloralkali process, and there 15 is no need to isolate them to prepare sodium hypochlorite. Hence NaOCI is prepared industrially by the electrolysis of sodium chloride solution with minimal separation between the anode and the cathode. The solution must be kept below 40 °C (by cooling coils) to prevent the formation of sodium chlorate.

The commercial solutions always contain significant amounts of sodium chloride (common salt) 20 as the main byproduct, as seen in the equation above.

Household bleach sold for use in laundering clothes is a 3-6% solution of sodium hypochlorite at the time of manufacture. Strength varies from one formulation to another and gradually decreases with long storage.

A 12% solution is widely used in waterworks for the chlorination of water and a 15% solution is 25 more commonly used for disinfection of waste water in treatment plants. High-test hypochlorite (HTH) is sold for chlorination of swimming pools and contains approximately 30% calcium hypochlorite. The crystalline salt is also sold for the same use; this salt usually contains less than 50% of calcium hypochlorite. However, the level of "active chlorine" may be much higher.

A weak solution of 1 % household bleach in warm water is used to sanitize smooth surfaces 30 prior to brewing of beer or wine. Surfaces must be rinsed to avoid imparting flavors to the brew; these chlorinated byproducts of sanitizing surfaces are also harmful.

US Government regulations (21 CFR Part 178) allow food processing equipment and food contact surfaces to be sanitized with solutions containing bleach provided the solution is allowed to drain adequately before contact with food, and the solutions do not exceed 200 parts 35 per million (ppm) available chlorine (for example, one tablespoon of typical household bleach containing 5.25% sodium hypochlorite, per gallon of water). If higher concentrations are used, the surface must be rinsed with potable water after sanitizing.

A 1 in 5 dilution of household bleach with water (1 part bleach to 4 parts water) is effective against many bacteria and some viruses, and is often the disinfectant of choice in cleaning surfaces in hospitals (Primarily in the United States). The solution is corrosive, and needs to be thoroughly removed afterwards, so the bleach disinfection is sometimes followed by an ethanol disinfection. Chlorine products can be corrosive to plant and equipment, people and is also costly.

Sodium hypochlorite is a strong oxidizer. Products of the oxidation reactions are corrosive. Solutions burn skin and cause eye damage, particularly when used in concentrated forms. However, as recognized by the NFPA, only solutions containing more than 40% sodium hypochlorite by weight are considered hazardous oxidizers. Solutions less than 40% are classified as a moderate oxidizing hazard (NFPA 430, 2000). There are numerous report s and 1 5 scientific papers discussing the problems associated with the use of chlorine. For example, the EPA in the 1990s raised skin absorption of chlorine to its top 10 carcinogen watch list, a professor of water chemistry at the University of Pittsburgh claimed that exposure to vaporized chemicals in the water supply through showering, bathing and inhalation was 2100 times greater than through drinking the water.

During the mid1970's monitoring efforts began to identify widespread toxic contamination of the nation's drinking water supplies, epidemiological studies began to suggest a link between ingestion of toxic chemicals in the water and elevated cancer mortality risks. Since those studies were completed a variety of additional studies have strengthened the statistical connection between consumption of toxins in water and elevated cancer risks. Moreover, this 25 basic concern has been heightened by other research discoveries.

"Chlorine is used almost universally in the treatment of public drinking water because of its toxic effect on harmful bacteria and other waterborne, disease-causing organisms. But there is a growing body of scientific evidence that shows that chlorine in drinking water may actually pose greater long-term dangers than those for which it was used to eliminate. These effects of 30 chlorine may result from either ingestion or absorption through the skin. Scientific studies have linked chlorine and chlorination by-products to cancer of the bladder, liver, stomach, rectum and colon, as well as heart disease, arteriosclerosis (hardening of the arteries), anemia, high blood pressure, and allergic reactions. There is also evidence that shows that chlorine can destroy protein in our body and cause adverse effects on skin and hair." "The presence of chlorine in water may also contribute to the formation of chloramines in the water, which can cause taste and odor problems." The use of chlorine and sodium hypochlorite in their presently known form as sanitizers therefore poses serious problems to the public. 6 Object of the Invention It is therefore an object of the invention to go some way in providing a useful and safe biocide or to at least provide the public with a useful choice.

Summary of the Invention The invention provides a process for the generation of carbon dioxide in solution in which the resulting chloride dioxide solution is stable.

The chlorine dioxide solution is preferably stable for up to 14 months.

The invention also provides a stabilized chlorine dioxide solution. The solution is preferably stable for at least 14 months.

The invention also provides a method of using the stabilized chlorine dioxide solution. The 20 solution is preferably stable for at least 14 months.

Surprisingly, the invention provides a unique process for producing a stabilized chlorine dioxide solution in which the presence of a certain amount of chlorite ion (CI02"-) in the aqueous medium helps stabilize the presence of CI02 in that solution.

The Cl02 may be either: added to the CI02 solution after it is formed; be residually present from incomplete oxidation of a CI02 - solution to CI02; or result from the initial degradation of a pure CI02 solution, where some of the CI02 is reduced 30 back to CI02"-.

The chlorine dioxide solution according to the invention has numerous uses. The product may be packed in a cardboard outer in which is contained a plastic "jerry can" containing the salt and a smaller "pottle" containing further salts. The Gross weight is 2.3 kilograms and measures .135 35 x. 135 x.200.

The contents make four hundred litres of usable product.

The product may be activated using the following procedure: obtain a suitable container normally a two hundred litre drum; preferably 500 grams of the salt is poured into water and agitated to dissolve it; 40 once dissolved, 500 mis of hydrochloric acid is added; 7 as the drum fills, 10 grams of salt may be taken from the pottle and to this may be added 500 mis of acid and 500 mis of water; and the mixture is added to the drum and allowed to fill.

The uses may include any of the following: 1. Water treatment 1:5000 to 1:15000 ration of active to water Depending on the measured or perceived level of contamination of the water source. 2. Disinfectant 1:100 which insures log 5 reduction of major contaminants in under thirty seconds. 3. Field Use 1 5 A bowser of fifty thousand litre capacity is driven to a pond. The water is considered to be of medium level contamination. The bowser is filled to near capacity and five litres of the chlorine dioxide is added. The water is then safe for human consumption.

A field kitchen needs sanitation. A solution of one part of chlorine dioxide to one hundred parts of water is made up. The resultant diluent is used as a hard surface sanitiser.

There is perceived to be an odour problem. A diluent as in above is made and the area is sprayed.

Corpses may be treated with chlorine dioxide to delay the effects of bacterial invasion postmortem. This matter has been discussed with Messrs. Mortech.

The uses for this product in all fields of sanitation are remarkable. It may be used as a mouth 25 wash, as a fungicide as an antiseptic on cuts and it does not have the inherent health risks associated with chlorine.

From the perspective of ease of cartage and manufacture there is no need for disposal considerations as the packaging may simply be burnt.

A complete assessment of chlorine dioxide regarding toxicity etc. is available for determination 30 on request. 4. Treatment of ground water The general procedure for treating ground water is: use antiseptic pumping equipment; introduce CI02 at the storage tank using a metering device; treat the water directly; 8 dosage depends on the bacterial loading. (It could range from 0.3 mgs/L to 1 mg per litre); for normal circumstances preferably use 0.3 to 1 mg per litre. For bacterial content of 100 coliforms per 110 mis of water preferably use 0.5 mg/L; after treatment, filter the water to rid it of impurities; store in hermetically sealed container; preferably, dosage is done on a weekly basis if the seal is not perfect; and this water is fit for human consumption.

. Health The following are some of the areas where chlorine dioxide in solution according to the 1 5 invention has proven effective: acne; athlete's foot; anti-cross infection; amalgamated infections; comedones; condyloma; dandruff; dermal damage; eczema; psorisis; fungus Infections; herpes simplex; muscle damage; scabies; and tendon damage (Soak for ten to fifteen minutes with a solution of one to twenty or one to forty). 6. Oral hygiene The product according to the invention is effective against: colibacillus; golden staphylococcus; white oidiomycetes; and for prevention of halitosis.

Halitosis is caused by microbes that can decompose thiamine acid, protein, peptone and non-vital epidermal cells into sulphides (H2S, CH3S. (CH2)2S. Gargling with 0.005% to 0.2 % solution promptly decreases 50 to 50% of volatised sulphides.

Extrasomatic tests show it kills the main pathogenic bacteria that cause dental caries, e.g. 99% 30 min S. Mutans.

It is effective against anaerobic bacteria.

Further tests show that it is efficacious against actinomycetes of gingivitis, cocci, spirochetes 35 caused by gingivitis, peridotites and gum bleeding. 7. Cleaning of artificial teeth Gargle or soak in solution of 1 to 200 40 8. Eve care The product can be used in the sterilisation of contact lenses. Apply directly. The low dosage means it is harmless, non-toxic and does not irritate the eye; 9 Conjunctivitis, use 5mg/L three times a day. Effective cure in three to five days; and the product is effective against styes, blood shot eyes etc. 9. Aguaculture Primarily for sterilisation, antisepsis and the increase of oxygen in the water.

The dosage is safe and non-toxic to shrimps, prawns, fish and shellfish. The pharmacodynamic time is one dosage effective for 10 to 15 days.

The effect is to increase the water quality by oxidation when acting as a bactericide. It oxidises 1 5 sulphides. Cyanide etc, inorganic compounds, chloro-phenols, thio and 2-tertiary amines and organic compounds that are harmful to shrimps and fish.

New bionomic oxygen is produced in the pond increasing the amount of dissolved oxygen. It effectively decreases the chemical oxygen consumption and values of ammonia and nitrogen in 20 that environment.

Infectious bacteria, viruses and harmful algae are promptly killed in the pond. Prevents and cures all fish diseases.

Dosages in this area would preferably be in the region of 1500 to 1600 ml per cubic metre, evenly distributed.

. Stockbreedinq Sterilisation, antisepsis and disease prevention. 11. Mushroom growing Sterilisation and antisepsis. 12. Animal Husbandry The chlorine dioxide solution according to the invention kills the various bacterial breeding units, bacteria spores, viruses, pathogenic micro-organisms and their carriers i.e. spores, Helminth, algae etc.

The product is able to treat and prevent foot and mouth disease, porcine erysipelas, porcine 40 pnuemopathy and other diseases caused by anthracoid spores, porcine viruses etc.

Removes odours and keeps a clean environment in sheds etc. The solution can be used as a spray or used to fumigate. 13. Fruit and vegetable post harvest Sterilisation and antisepsis is achieved by dipping and washing. Bacteria and fungi are destroyed. Any remaining pesticides are destroyed; the nett effect is to extend the shelf life of the product. 14. At home Removing odours in the refrigerator, place a solution in a bowl inside the fridge; Toilet cleaning - directly into the bowl; Dermatophytosis (Smelly Feet), wash feet and socks in solution. 15. Hospital and medical Instruments Sterilisation and antisepsis Rinse; Wards Sterilisation and antisepsis Fumigate Removes odours; and Sewage Sterilisation and antisepsis Treat Direct. 16. Miscellaneous applications Odour Abatement; Fumigation; Food and Beverage Industry; Purifying water, cleaning plant and equipment, achieves sterilisation and antisepsis, apply as circumstances dictate; Marine and Meat Products; Shelf life extension and water quality, plant and equipment cleaning, Odour abatement; Water Circulation Systems Sterilisation, Iron and Manganese removal, algae control, apply directly to water 30 Petroleum Industry; and Sterilisation, Iron and Manganese removal, algae control and bacteria control, apply directly to water. 17. Weaving, paper making, printing and dyeing industries 35 Colour removal and Bleaching. 18. Sewage treatment Water Treatment in the Chemical, Textile, Papermaking and Dyeing Industries, apply direct to water. 40 19. General food industry Sterilisation of work areas, conveyors, pipelines, transport, drinking water, tools, plant and equipment, working clothes, masks and head gear, spray or soak with 1 to 400 or 1 to 600 solution; and 11 Hotels Restaurants and Food Preparation Industries, all hard surfaces, spray or soak with 1 to 200 solution.

. Around the Farm Item Concentration Method Drinking Water 1 5000 - 1:10000 Add directly to water Poultry Shed 1 200- 1:500 Soak equipment for 5 mins Milk Inhaler 1 200- 1:500 Wash and rinse Teat Disinfectant 1 200- 1:500 Wash or spray direct Milk anti-corrosive 1 2000 - 1:5000 Add as per rate Disinfecting pipes 1 500 Wash and Flush Animal hooves 1 200- 1:500 Soak and Wipe Working clothes 1 500 Soak pre wash Various containers 1 500- 1:1000 Clean and sanitise Brief Description of the Drawings The invention will now be described in detail with reference to the following drawings in which 1 5 Figure 1 shows an apparatus for the preparation of a chlorine dioxide solution; Figure 2 shows a graph of the ability of the chlorine dioxide solution to kill micro-organisms in fluids.

Detailed Description of the Invention The applicant has found that aqueous CI02 solutions degrade in the following manner: 2 CIO 2 +H 2 O+CIO 2" - +CIO 3 +2H + Although acidic solutions suppress the degradation, it is largely complete even in fairly acid environments.

The applicant has found that the presence of a certain amount of chlorite ion (CIO 2" in the aqueous medium will help stabilise the presence of CI02 in that solution.

This CIO 2 '- may be either a) added to the CI02 solution after it is formed; b) be residually present from incomplete oxidation of a CI02~- solution to CI02; or c) result from the initial degradation of a pure CI02 solution, where some of the CIO 2 is reduced back to CIO 2"-. 12 The basis for the surprising stability of the CI02 in the presence of CI02 ion is putated to derive from the existence of a bimolecular charge-transfer complex involving one molecule each of CI02and CI02", as follows: Q=1.6 mol"1 CIO 2 +CIO 2" -> [Cl204 ]" - Thus, in solutions that contain both CI02 and CI02", it can be expected that a portion of the CI02 10 will be tied up in complex form, and not be available per se as free CI02. However it should be also noted that the oxidation potential of [CI 204]is reportedly higher than that of CI02, so that CI02 solutions also containing CI02 , and therefore the complex, ion would be expected to have a greater oxidation capacity than might be expected from simply that calculated from the level of Cl02 present. This increased capacity would be expected to be associated with, for 15 example, greater disinfection or a greater ability to destroy oral malodorants than a comparable CI02 solution with no additional chlorite present.

On the basis of the above data, and the theory underlying the need for a specific minimum amount of CI02"- ion to be present with respect to CI02 in order for CIO 2 to achieve a certain level of stability in the aqueous solution, the molar ratio of CI02"-: CI02 preferably should be at 20 least 1:1, but not more than about 20:1. Above that relative amount of chlorite ion with respect to chlorine dioxide, a significant generation of Cl02from the CI02" - will tend to create a desired increase of CI02 in the aqueous solution over a period of time, rather than maintaining a fairly constant level.

Stability Testing The following test was used to analyse the sample.

Two methods of testing stability have been employed.

Apparatus for the preparation of a chlorine dioxide stock solution I: The entire apparatus must be set up in a fume cupboard. 1. Connect the inlet of a 500-ml gas-washing bottle, filled with 100 ml of water GR, to a pure-air source or a compressed-nitrogen cylinder fitted with a pressure-reduction manometer. 2. Connect the outlet of this 500-ml gas-washing bottle with a PE tube to a gas-distribution tube fitted with a joint adapter into the left ground joint of a 500-ml three-necked flask that is standing on a magnetic stirrer, inserting the gas-distribution tube all the way to the bottom of the three-necked flask. Fill the three-necked flask with 100 ml of water GR. Place a 100-ml dropping funnel with a Teflon cock plug and a pressure-relief tube in position on the middle ground joint of the three-necked flask. 3. Connect the right ground joint of the flask to the inlet of a 500-ml gas-washing bottle. Fill 40 50 ml of the 1 % sodium chlorite wash solution into this bottle. 4. Connect the outlet of this gas-washing bottle to the inlet of a 1000-ml gas-washing bottle, fitted with a sieving frit, containing 500 ml of water GR. The 1000-ml gas-washing 13 bottle serves as an absorber unit for the chlorine dioxide and must be cooled externally with iced water. Refer to Figure 1.

Preparation of a chlorine dioxide stock solution I: To prepare the chlorine dioxide stock solution I fill 10 g of sodium chlorite for synthesis, 250 ml 10 of water GR, and a magnetic-stirrer rod approximately 2 cm long into the 500-ml three-necked flask fill 25 ml of sulfuric acid 25% GR into the dropping funnel and close the funnel with a suitable ground-glass stopper. Stirring and the slow, dropwise addition of the sulfuric acid set off the development of the gaseous chlorine dioxide in the three-necked flask. Refer to Figure 1. 1 5 The gaseous chlorine dioxide is expelled by blowing pure air or nitrogen as the carrier gas through the apparatus, in which process the gas-washing bottle containing water GR to the left of the three-necked flask serves as a bubble gauge. The gas-washing bottle to the right, filled with 50 ml of the 1 % sodium chlorite wash solution, removes any traces of chlorine that may be present as a result of the formation of chlorine dioxide from sodium chlorite. The chlorine 20 dioxide is collected or absorbed in the 1000-ml gas-washing bottle containing 500 ml of cooled water. The rate of flow of the carrier gas must be metered in such a way to ensure that the formed chlorine dioxide is promptly expelled. The stock solution I (from the 1000-ml gas-washing bottle), prepared according to the above schedule, can contain 250-600 mg/l of chlorine dioxide.

The sulfuric acid set off the development of the gaseous chlorine dioxide in the three-necked flask. Refer to Figure 1.

Titrimetric assay of the chlorine dioxide stock solution I: In a 250-ml conical flask with a ground-glass stopper mix 2 g of potassium iodide GR, 50 ml of water GR, and 2 ml of sulfuric acid 25% GR. Into this solution pipette 25 ml of the chlorine dioxide stock solution I using a volumetric pipette. Leave the mixture to stand in the closed flask for 5 minutes in the absence of light. Titrate the released iodine with sodium thiosulfate solution 0.1 mol/l against zinc iodide-starch solution GR as the indicator. The colour changes from blue 35 to colourless. Make a note of the amount of sodium thiosulfate solution 0.1 mol/l consumed in the titration step.

Notes regarding pipetting with the volumetric pipette: To pipette the chlorine dioxide stock solution I, when expelling the pipette contents it is important always to insert the tip of the volumetric pipette in the solution previously filled into the 40 conical flask as a measure to minimize any loss of the analyte.

Calculation: mg chlorine dioxide per ml stock solution I = A x N x 13.49ml sample 14 A = Consumption of sodium thiosulfate solution 0.1 mol/l N = Normality of the sodium thiosulfate solution 0.1 mol/l = 0.1 The chlorine dioxide stock solution I prepared in this manner is used to prepare diluted working solutions (e.g. 100 mg/l). These dilutions must be used immediately, since they remain stable for a maximum time of one hour in the closed volumetric flask.

Example for the preparation of a working solution of 100 mg/l chlorine dioxide: It has, for example, been titrimetrically calculated that the chlorine dioxide stock solution I has a concentration of 1.25 mg chlorine dioxide per ml. The following formula is employed for the preparation of a 100-mg/l chlorine dioxide working solution: 100 /1.25 = 80 In other words, 80 ml of the 1.25 mg/ml chlorine dioxide stock solution is measured into a 100-1 5 ml volumetric flask with a buret and made up to the mark with water GR. The concentration is now 100 mg/l chlorine dioxide.

Results It is a constant feature of the literature that if chlorine dioxide in aqueous solution away from UV light and under 30 degrees Celsius will have a long shelf-life. Secondly it was found various 20 plastics were more accommodating of chlorine dioxide than others. The literature pointed to HDPE.

Therefore a batch was prepared using the system described in Chemistry and Manufacturing p.17.

The batch was tested for the concentration of chlorine dioxide using US-Standard Methods AWWA, APHA, WCPF 17th Edition (1989) described above the results are printed in Table 1 below. The results showed stability based on a 96 day trial.

Two further samples from the original batch were taken one was packed in an amber coloured PET bottle and the second in a HDPE plastic pouch.

Both were placed in an area out of direct sunlight and further the second sample was protected from light by a cardboard box.

Measurements of their voltage were taken using an ORP meter in accordance with the testing procedure described in the ORP related articles above.

The results are printed below in Tables 1, 2, and 3. 40 The trials were discontinued at 96 days, 9 months and 14 months.

If the solution were to kept and below 30 degrees Celsius and out of direct sunlight then a safe shelf-life would be about 12 months.

Table 1 Storage Stability Test - Chlorine Dioxide Test Level Air Vol Found Taken Statistical Analysis 0.13ppmCI02 (L) ppm ppm n Mean Std Dev CV Recovery (%) Day 1 116 0.133 0.130 112 0.128 0.130 116 0.128 0.130 117 0.126 0.130 119 0.115 0.130 104 0.127 0.130 Day 5 116 0.125 0.130 112 0.122 0.130 116 0.117 0.130 117 0.123 0.130 119 0.125 0.130 104 0.118 0.130 Day 15 116 0.133 0.130 112 0.129 0.130 116 0.127 0.130 117 0.125 0.130 119 0.131 0.130 104 0.157* 0.130 0.126 0.006 0.047 97.1 0.122 0.003 0.028 93.6 0.129 0.003 0.025 99.2 Day 30 116 0.126 0.130 16 112 0.130 0.130 116 0.130 0.130 117 0.128 0.130 119 0.125 0.130 104 0.161* 0.130 Day 48 116 0.131 0.130 112 0.131 0.130 116 0.127 0.130 117 0.128 0.130 119 0.127 0.130 104 0.164* 0.130 Day 96 116 0.137 0.130 112 0.132 0.130 116 0.128 0.130 117 0.133 0.130 119 LIA 0.130 104 0.161* 0.130 0.128 0.002 0.018 98.3 0.129 0.002 0.016 99.1 0.133 0.004 0.028 102 LIA = Lost in Analysis * Outlier - not used in statistical analysis ORP STABILITY TESTS The second method used to prove stability is that of Oxygen reduction potential.

ORP technology has been gaining recognition worldwide and is found to be a reliable indicator of bacteriological water quality for sanitation - determine free chlorine parameter. In swimming 17 pool application, the ideal ORP value is approximately 700 mV where the Kill Time of E.Coli bacteria is the fastest to ensure good water quality.

As can be seen from the results shown I Figure 2,ORP indicates that most micro-organisms are killed in fluids in excess of 650 mV. Our results show that the ORP level of our product is 10 constantly above 900mV. Samples were kept in an office environment in Richmond on an exposed bench.

Sampling Equipment EUTECH INSTRUMENTS Waterproof ORPTestr 10 1 5 Table 2 Sample PET bottle Date / Year 2005 mV April 20 975 May 10 980 May 20 971 June 12 970 June 21 965 July 10 960 July 20 954 Aug 8 926 Aug 24 960 Sept 5 955 Sept 20 954 Oct 12 948 Oct 28 941 Table 3 Card Board Wine Cask Date / Year 2005 / 2006 mV July 7 970 July 18 980 August 3 987 August 24 1143 September 5 1135 September 20 1130 October 21 1036 November 29 977 January 18 1021 March 14 1008 April 20 991 18 May 11 970 June 2 970 July 12 975 Fruit and Vegetable Industries For many years fresh produce industries have been searching for an effective ready to use sanitiser that rapidly destroys all types of microorganisms and also provides maximum employee and environmental safety.

Likewise horticultural operations have been seeking broad-spectrum ecocides without harmful residuals or long lasting withholding periods.

ONE PREFERRED EMBODIMENT - PREPARATION OF S1000 15 TO MAKE 200 LITRES Ingredients are marked either "A" (sodium chlorite), "B" (hydrochloric acid) and "C" (sodium chlorite) - it being surprisingly found that the order of mixing of said components being essential to providing a unique solution of chlorine dioxide that has the surprising advantage of affording a sanitiser which remains stable over hitherto unimagined periods of time.

The unique method and resultant end product leads to reduced wastage of raw materials, a serious saving of time and resources, and an end product which satisfies a long-felt want in the marketplace. 1. take 500 grammes of "A" and add to 198 litres of water 2. wait for five minutes for "A" to dissolve 3. add 500 mis of agent "B" 4. add 1 litre of 30 to 32 % hydrochloric acid to 1 litre of water. (always add acid to water) . take 20 grammes of agent "C" and add to acid and water - a reaction will take place resulting in bubbling, heat and the giving off of a yellowish green gas. 6. when reaction is under way pour into holding vessel 7. screw down tops 40 The inventor has experimented with the process and has come up with the following variation: 19 Steps 1 and 2 remain the same Step 3 changes. Rather than reacting the compound in the acid/water diluent one variation is to now add the necessary amount of compound C into the container (without reacting it) THEN -ADD THE ACID/WATER MIX This makes for a better reaction and a safer one as one is not exposed to the gas as it is made. The draw back is that the reaction is slower and the finished goods must be left overnight for the reaction to take place completely. 1 5 Nevertheless, the process is safer and also allows for a stronger concentration of the active. What it means of course is that this will necessitate a certain amount of pre-planning as makeup cannot be left to the last minute.

Where can Chlorine Dioxide be used Post harvest sanitation of fruit and vegetables surface through flume wash to improve shelf life and freshness.

Removal of unwanted human pathogens on the surface of fruit and vegetables including E. coli and Listeria.

No rinse sanitation of equipment used to harvest produce.

Disinfection of flume and process waters including dump tanks and spray lines.

Sanitation of hard surfaces.

Reduction of pathogen load of amongst others: Alternaria Aspergillus Botrytis Cladosporium Colletotrichum Cylindocarpon Downey Mildew Erwina European Canker Fusarium Pencillium Phoma Phytophora Powdery Mildew Drench Washing Washing of the produce is undertaken in baths. This wash water is responsible for removing mainly soils off the produce. Hence microbial loading of the water increases, thereby offering a contamination vector of the other produce. It is therefore essential to treat this wash water with a disinfectant in order to improve and control the microbial quality of the water. In this way, one is able to offer some surface microbial reduction on the produce, thereby extending the shelf life.

When looking at reductions in counts there 3 are factors that determine the efficacy of the disinfecting solution: contact time, concentration and turbulence (turbulence within wash 10 solutions). The shorter the contact time and the absence of turbulence require a higher concentration of Chlorine Dioxide.

Therefore, if washing of produce is under taken in a proper bath where there is water turbulence that drives the produce through the packing line. The recommended dosage is 250 - 500ml to 1 1 5 Litre of Chlorine Dioxide per 1000 Litres water, with a contact time of 1 minute.

Washing of Deciduous Fruit Deciduous fruit is washed in dump tanks or spray units with a disinfectant in order to inactivate the spores of the post harvest fungal diseases and to reduce bacterial contamination in wash 20 waters from a food safety perspective. In many instances the water used is of poor quality in that it contains suspended solids, organics and is microbiologically contaminated. These factors complicate the requirements of meeting the customer's need of high quality fresh produce with no spoilage.

DOSAGE 250ml to 500ml Chlorine Dioxide per 1000 Litres of water Washing of Citrus Fruit Citrus is washed in dump tanks or high pressure spray units with a disinfectant in order to 30 inactivate the spores of the post harvest fungal diseases and to reduce bacterial contamination in wash waters from a food safety perspective. In many instances the water used is of poor quality in that it contains suspended solids, organics and is microbiologically contaminated. These factors complicate the requirements of meeting the customer's need of high quality fresh produce with no spoilage.

Citrus Dosage 1 Litre Chlorine Dioxide per 1000 litres of water Potatoes 40 Dose Potato dipping tank 2- 4 Litres per 10OOIt of water Product should topped up when dipping tank has lost 10% of tank volume Dipping tank mix should be replaced every 50,000kg of seed.

Plant and Machinery should be disinfected every before use 21 Sprayed with Knapsack - 400ml per 20L of water Hydroponics Hydroponics or intensive farming needs strict bio-security control to eliminate the various vectors that can be used to spread disease in a hydroponics facility. We need to focus on each 10 aspect to reduce the potential for the spread of disease by.

Treatment of fertigated water (fertiliser, nutrient and biological control agent (BCA) mixes) to prevent the spread of root diseases such as pythium, fusarium, phytophora, alternaria and rhizoctonia microorganisms. This is particularly necessary where nutrient gravel film systems 15 are used where the fertigated water is continually re-cycled or where regulations require the fertigated water to be re-cycled. Dosage: 40 ml to 100 ml of Chlorine Dioxide per 1000 Litres of water. (2 L to 5 L per 50 000 Litres of water).

Dry Packing (I.e. Lettuces That Are Packed Whole) When produce is packed without washing, spraying with Chlorine Dioxide onto the produce, especially onto the cut ends and damaged areas, extends the shelf life. This will impact on the shelf life by reducing oxidative browning and microbial rot of produce. Chlorine Dioxide can be applied as a very fine spray onto the produce (do not wet the produce); this is done at a dosage of 2.5 L Chlorine Dioxide per 1000 Litres of water.

Hydro cooling Of Produce Chlorine Dioxide has been successfully used in the hydro cooling of vegetables as it can inactivate microorganisms at refrigeration temperatures. The typical dosage is 1 L of Chlorine Dioxide to 1000 litres of water. We have found that not only do we keep the produce free of 30 fungal contamination but the copper coils are also kept clean during the cooling cycles as well.

The list of vegetables, which have been treated, include, amongst others: Beans; Carrots; Celery; Ginger; Lettuce; Melons; Onions; Okra; Peas; Parsnips; potatoes; Sweet potatoes; and Tomatoes.

Fruit and Vegetables Vegetables Vegetables of all kind are washed, cut and packed (e.g. in plastic bags). 40 Customers are supermarkets and fast food producers.

Previous treatment Usually Chlorine is used for microbiological control with concentrations varying between 100 - 200 ppm. 22 Problems with previous treatment Chlorine created a smell problem during processing in the processing hall with operators complaining of eye and skin irritation. pH very often above 7.5 where microbiological treatment is often not effective with chlorine.

Batch washing, Case studies Water change every 6-8 hrs. typical dosage: 6 ppm 1 5 Spraying, typical concentrations Onion rings 6 ppm Carrots 1 ppm Benefits of chlorine dioxide Shelf life increased by factor 3 Smell problem decreased significantly.

Washing of cut lettuce. - Quality requirements: Salmonella zero 25 Listeria zero E.Coli zero.

Must pass sensory evaluation test criteria (no chlorine taste).

Appearance of lettuce must be good.

TPC must be within guidelines at day 10.

TPC = Total Plate Count (microbiological surface contamination) Description of old chlorine disinfection system.

Chlorine at 100-200ppm. Dosed using sodium hypochlorite 12.5% Terrible chlorine smell in factory with workers complaining of eye and skin irritations.

Impossible to control chlorine residual and required manual chemical addition every 15 minutes pH control not possible as always creeping high.

Required to dump a lot of water to maintain chlorine residual which was high cost for chilling and extra ice 40 E.Coli was not always zero.

Always concerned about Listeria as Listeria not affected by chlorine at low temperatures TPC at day zero was inconsistent usually 1 x 105, 3 x 105 and occasional 1x10® counts Description of CI02 system 23 Chlorine dioxide at 1 .Oppm in 2 stage wash. First wash stage is 8 deg. C and second wash stage is 2 deg. C.

Dosing is done automatically and automatic residual control.

No chemical smell in the factory at all.

Operators do a check on the dosing equipment every hour or so but do not add any chemicals manually. pH is automatically controlled to 7.5.

Very little dumping of water and only chilled water is used. Chemical running cost is very low. E.Coli is always zero. 1 5 No concerns about Listeria as CI02 will easily kill Listeria at low temperatures.

TPC at day zero is consistent and always less than 7 x 104 Producer of frozen corn cobs, kernels and peas.

General process water contains 0.5 ppm chlorine (town supply).

Process description General process water contains 0.5 ppm chlorine (town supply).

Wash water is process water with 2 ppm chlorine dioxide added by metering device.

Corn is blanched and then cooled down. As the corn is cooling, microbiological growth can 25 occur.

The corn is cooled by water spraying with 2 ppm chlorine dioxide (critical stage).

Advantages/Benefits: No taste and odour influence on the corn.

CI02 works well in an environment of high organic loading.

No chlorine smell in the factory hall.

Easy generation, dosing and control of disinfection.

Processing Of Spinach 35 Processing steps: Spinach is moved dry (removing of beetles and caterpillars) Washed with cold tap water Blanching at 80°-90°C, cooling The water from the last blanching segment in taken to a cooler 40 Production Two processing lines, each 12 T/hr Make-up water per line 12 m3/hr 24 Dosing of CI02 In the cooler CI02 is dosed, time proportional, intercooled with the last zone of the washing machine, dose: 100 g/hr Processing of Tomatoes Tomatoes are brought to the processing factory by truck and then transported by flume to the tomato paste production area. Chlorine Dioxide is used to destroy moulds on the tomatoes and in the flume tank.

Processing steps: Tomatoes are dumped from the truck onto a conveyor.

Coarse rinse with town water sprays to remove dirt and stems, leaves etc.

Tomatoes fall into flume tank (20 m3). The flume water is pumped to the sorting conveyor and back in a closed circuit with the tomatoes. Operators remove unacceptable product.

Make-up condensate water is continually added (5 m3/hr) from the tomato paste process. 20 Chlorine Dioxide is dosed into the flume water to maintain concentration of 0.2 - 0.4 ppm of CI02. pH of the flume water goes to 4.0 and this is not corrected as it is acceptable.

Method of concentration control: Directly into flume. By-pass water is the condensate flow.

Control to 650mV This system is only used in wet weather and occasionally during dry weather. Mould is a bad problem when there is a lot of rain during harvest.

Previous Treatment Used sodium hypochlorite and due to the high concentration of organic material in the fiume water, had difficulty maintaining any free chlorine residual. This meant that moulds were not controlled and surfaces were fouled. In addition, operators would occasionally stop work due to chlorine smell in the sorting area.

Advantages I benefits Low concentration of chlorine dioxide is very effective in destruction of moulds on the tomatoes. These moulds would negatively affect the past production process if present.

Low concentration of chlorine dioxide is very effective in destruction of moulds in the flume 40 water. If untreated, the moulds attach to surfaces of tanks and flumes and look like "meat". Eventually, they foul screens and smell.

Chlorine dioxide effective at pH 4.0 No smell for operators Very low running costs No chlorinated organic by-products.

Possible build up of chlorite in the flume tank can affect the skin of operators when they handle the tomatoes i.e. hands, arms and face. If they wear gloves then this can be of help.

Processing Of Potatoes Potatoes are brought to the processing factory by truck and placed into heaps. They are then washed and cut into french fry shapes prior to freezing. Water used for processing is from a dam. It is flocculated and disinfected with chlorine dioxide. 1 5 Processing steps: Potatoes are cut and washed with chlorine dioxide treated water.

Chlorine Dioxide is flow pace dosed into the treated water at a dose of 1.0 ppm to maintain concentration of 0.5 ppm of CI02.

Method of concentration control: Dosing directly into treated water Previous Treatment Previously used sodium hypochlorite and due to the high concentration of organic material in the 25 dam water, had difficulty maintaining any free chlorine residual into factory. Processed product was developing an unusual taint. Chlorine dioxide treatment removed the taint.

Advantages/ben ef its: Chlorine dioxide dose at 1 ppm was a better micro-biological control agent than chlorine at 5-30 10ppm.

No product taint.

Automatic operation simple and effective Very low running costs No chlorinated organic by-products.

Processing Of Citrus Washing stage: Immersed in water containing chlorine dioxide.

Aim is the reduction of: Geotrichum Candidum Sour Rot Spores; 40 Penicillium Digitatum blue mould; and Green mould.

Results: 2 ppm CI02 dosage 26 0.35 ppm CI02 residual Dosage are controlled via redox as wash water is very dirty.

Wash water temperature 20 deg.C, pH 8 Outturns significantly less with CI02 than previous Nylate (bromine) or chlorine treatment No taste or odour problems with the oranges.

Shelf life increased threefold.

Elimination of need for fungicide Other Issues Exhaust system was necessary for removal of excess CI02 and airborne spores.

A "Food Stock" Manufacturer Processing steps: Onions are cut and fried on a hot plate resulting in complaints regarding cooking odours in industrial area.

Continuous fog of Chlorine Dioxide into extraction hood mixed at 5 Litres per 100 Litres of water and fogged at 3.5 Litres per Hour.

Method of concentration control Dosing directly into treated water Previous Treatment None Advantages/benefits: Odours eliminated No product taint.

Automatic operation simple and effective Very low running costs No chlorinated organic by-products.

An Apple orchardist Packs fruit for local markets in a year round operation. Water in the flume gets very discoloured and malodorous from decayed fruit ex cool store and CA storage. Flume and water dump approximately 14,500 Litres capacity. 40 Processing steps: Shock dose 10 Litres Chlorine dioxide.

Add 3 Litres each week when "topping-up" water level. 27 Method of concentration control: Dosing directly into treated water Previous Treatment: Chlorine.

Advantages/benefits: Water visibly clearer and not malodorous.

No product taint No product taint.

No fermentation of pulpy fruit in the waste bins Simple and effective.

Very low running costs No chlorinated organic by-products.

Client intends to drench apples, pears, peaches prior to cool storage to prevent spoilage 20 organisms infecting stem punctures etc.

A Medical equipment supplier Sales of new and used equipment plus Hire A special inflatable mattress returned from hire with bad smoke odours Processing steps: Wash with 1 litre Chlorine dioxide to 10 Litres of water.

Leave in shade for 30 minutes and allow to dry in air Method of concentration control: Dosing directly into water Advantages/benefits: Mattress completely odour free Mattress sanitised 35 No need to throw the mattress away.

No fermentation of pulpy fruit in the waste bins Automatic operation simple and effective Very low running costs No chlorinated organic by-products. 40 Fumigation SARD. (Specific Apple Replant Disorder) Disease Controls 28 For the control of soil borne fungal and bacterial pathogens Directions for use 1. Work ground to a fine tilth before rain. 2. Apply Chlorine Dioxide at a rate of 60 Litres per ha, plus an Organo -Silicone such as Rhino at 100mls/1 OOIt, 3. Apply a minimum of 500 Litres of water per ha. 4. Incorporate into soil by renovator immediately.

. Plant trees/plants into ground. 6. Individual plant applications should be made at 1L per 100L plus Organo-Silicone such as Rhino @ 100mls/1 OOIt Apply a minimum of 20 Litres of mixed product per planting hole.

A soil conditioner and/or nutritional supplement should follow all applications.

Bacterial pathogens isolated from raw vegetables Vegetable Country Pathogen Prevalence % Reference 40 Alfalfa U.S.A Aeromonas Callister (1989) Artichoke Spain Salmonella 3/25 12.

Ruiz et al. (1987b) Asparagus U.S.A Aeromonas Berrang et al.

Bean sprouts Malaysia L. monocytogenes 85 Malaysia Salmonella Arumugaswamy Sweden Salmonella Andersson Jong Thailand Salmonella 8.7 Jerngklinchan (1993) Beet leaves Spain Salmonella 4/52 7.7 Ruiz et al. (1987b) Broccoli Canada L. monocytogenes 13.3 Odumeru et al. (1997) U.S.A Aeromonas Berrang et al. (1989) U.S.A Aeromonas 5/16 31.3 Callister Agger Cabbage Canada L. monocytogenes 2.2 Schlech et al. (1983) Canada L. monocytogenes 1/15 6.7 Odumeru et al. (1997) Mexico E. coli 0157:H7 1/4 25.0 Peru Saudi Arabia Saudi Arabia Spain U.S.A V. chlolerae L. monocytogenes Y. enterocolitica Salmonella 7/41 C. botulinum 1/337 Zepeda-Lopez (1995) Swerdlow et al. Salamah (1993) Salamah (1993) 17 Ruiz etal.(1987b) 0.3 Lilly etal. (1996) 29 U.S.A L. monocytogenes 1/92 1.1 Heisick et al. (1989b) Carrot Lebanon Staphylococcus 14.3 Abdelnoor et al.

Saudi Arabia L. monocytogenes Salamah (1993) Saudi Arabia Y. enterocolitica Salamah (1993) Cauliflower Netherlands Salmonella 1/13 7.7 Tamminga et al.

Spain Salmonella 1/23 4.5 Ruiz et.al. (1987b) U.S.A Aeromonas Berrang et al. (1989) Celery Mexico E. coli 0157:H7 6/34 17.6 Zepeda-Lopez (1995) Spain Salmonella 2/26 7.7 Ruiz etal. (1987b) Chili Surinam Salmonella 5/16 31.3 Tamminga et al.

Cilantro Mexico E. coli 0157:H7 8/41 19.5 Lopez et al. (1995) Coriander Mexico E. coli 0157:H7 2/10 20.0 Lopez et al. (1995) Cress sprouts U.S.A B. cereus Portnoy et al. (1976) Cucumber Malaysia Pakistan L. monocytogenes 4/5 80 Arumugaswamy L. monocytogenes 1/15 6.7 Vahidy (1992) Saudi Arabia L. monocytogenes Salamah (1993) Saudi Arabia Y. enterocolitica Salamah (1993) U.S.A L. monocytogenes Heisick et al. (1989b) Egg plant Netherlands Salmonella 2/13 1.5 Tamminga Endive Netherlands 25 Fennel Italy Green onion Leafy veg.

Leeks 30 Lettuce Italy Salmonella 2/26 7.7 Salmonella 4/89 71.9 Canada Campylobacter 1/40 Malaysia Salmonella 1/24 4 Malaysia L. monocytogenes 22 22.7 Spain L. monocytogenes 20 Salmonella 82/120 Tamminga Ercolani 2.5 Park, Sanders 4 Arumugaswamy Arumugaswamy de Simon et al.

Vegetable Canada Canada Lebanon Netherlands Country Campylobacter 2/67 3.1 Park, Sanders (1992) L. monocytogenes 3/15 20 Odumeru et al. (1997) Staphylococcus 14.3 Salmonella 2/28 7.1 Abdelnoor et al. (1983) Tamminga et al. (1978) Pathogen Prevalence % Reference 40 Lettuce Saudi Arabia L. monocytogenes Saudi Arabia Y. enterocolitica Spain Mungbean Mushrooms U.S.A U.S.A U.S.A Mustard cress U.K.

Salmonella Aeromonas Salmonella C. jejuni 3/200 1.5 Salmonella Mustard sprouts U.S.A B. cereus Salamah (1993) Salamah (1993) Ruiz et al.(1987b) Callister, Agger (1989) O.Mahony et al. (1990) Doyle, Schoeni (1986) Joce et al. (1990) Portnoy et al. (1976) Canada Campylobacter 1/42 2.4 Park, Sanders (1992) Parsley Egypt Shigella 1/250 0.4 Satchell et al. (1990) Lebanon Staphylococcus 7.7 Abdelnoor et al. (1983) Spain Salmonella 1/23 4.3 Ruiz et al.(1987b) Pepper CanadaL. monocytogenes 1/15 6.7 Odumeru et al. (1997) Sweden U.S.A U.S.A Potatoes Saudi Arabia Saudi Arabia 15 Spain U.S.A U.S.A Canada Prepack salads N. Ireland 20 U.K.

U.K.

Andersson et al. (1989) Lilly et al. (1996) Callister, Agger (1989) Salamah (1993) Salamah (1993) Salmonella C. botulinum 1/201 0.5 Aeromonas L. monocytogenes Y. enterocolitica L. monocytogenes 2/12 16.7de Simon et al. (1992) L. monocytogenes 19/70 27.1 Heisick etal. (1989a) L. monocytogenes 28/132 21.1 Heisick et al. (1989b) Campylobacter 1/63 1.6 Park and Sanders (1992) L. monocytogenes 3/21 14.3Harvey, Gilmour L. monocytogenes 4/60 13.3 Sizmur, Walker (1988) L. monocytogenes Velani, Roberts (1991) Radish Lebanon Staphylococcus 6.3% Abdelnoor et al. (1983) Saudi Arabia L. monocytogenes Salamah (1993) Y. enterocolitica Salamah (1993) L. monocytogenes 25/68 36.8 Heisick et al. (1989a) Campylobacter 2/74 2.7 Park and Sanders (1992) L. monocytogenes 19/132 14.4Heisick et al. (1989b) Salmonella 1/250 0.4 Satchell et al. (1990) S. aureus 13/256 5.1 Houang et al. (1991) L. monocytogenes 6/15 40 Odumeru et al. (1997) Shigella 3/250 1.2 Satchell et al. (1990) Egypt S. aureus 3/36 8.3 Satchell et al.(1990) Spain Aeromonas 2/33 6.1 Garcia-Gimeno (1996a) Spain L. monocytogenes 30 Garcia-Gimeno.

U.S.A Staphylococcus Harris et al. (1975) Germany L. monocytogenes 6/263 2.3Breer (1992) N. Ireland L. monocytogenes 4/16 25 Harvey, Gilmour (1993) U.S.A C. botulinum 2/82 2.4 Lilly et al. (1996) U.K. Y. enterocolitica Brockelhurst (1987) Canada Staphylococcus 13/54 24 Prokopowich (1991) Soybean sprouts U.S.A B. cereus Portnoy et al. (1976) Spinach Canada Campylobacter Park and Sanders (1992) Spain Salmonella 2/60 3.3 Garcia-Villanova (1987b) U.S.A Aeromonas 2/38 5.2 Callister, Agger (1989) Saudi Arabia 25 U.S.A Canada U.S.A Egypt U.K.

Canada Egypt Salad greens Salad veg. 40 Seed sprouts 31 Sprouting seedU.S.A B. cereus 56/98 57 Harmon et al. (1987) Vegetable Country Pathogen Prevalence % Reference Tomato Pakistan L. monocytogenes 2/15 13.3 Egypt Salmonella 2/250 0.8 France Y. enterocolitica 4/58 7 France Y. enterocolitica 15/30 50 Salmonella 3/43 7 L. monocytogenes 7/102 6.9 Y. enterocolitica 1/102 1.0 L. monocytogenes 8/103 7.8 Iraq Italy Italy Spain Spain Vahidy (1992) Satchell et al. (1990) Catteau et al. (1985) Darbas et al.(1985) 7 Al-Hindawi (1979) Gola et al. (1990) Gola et al. (1990) de Simon (1992) Salmonella 46/849 5.4 Garcia-Villanova(1987a) Taiwan L. monocytogenes 6/49 12.2Wong et al. (1990) U.K U.S.A L. monocytogenes 4/64 6.2 Salmonella 4/50 8.0 MacGowan et al. (1994) Rude et al. (1984) Examples of pathogens associated with fruits and vegetables involved in outbreaks of food-borne disease Agent Implicated Suspected food Reference 40 Bacillus cereus Sprouts Campylobacter Cucumber Campylobacter jejuni Lettuce Clostridium botulinum Vegetable salad Cryptosporidium Cyclospora E.Coli 0157 E.Coli 0157 E.Coli 0157 E.Coli 0157 Fasciolia hepatica Apple cider Raspberries Radish sprouts Apple juice Apple cider Iceberg lettuce Watercress Giardia Vegetables, incl. carrots Hepatitis A virus Iceberg lettuce Hepatitis A virus Raspberries Hepatitis A virus Strawberries Norwalk virus Tossed salad Salmonella Agona coleslaw & onions Salmonella Miami watermelon Portnoy et al. (1976) Kirk et al. (1997) CDC (1998) PHLS (1978) CDR (1991) Herwaldt et al.(1997) WHO (1996) CDC (1996) Besser et al. (1993) CDR(1997) Hardman (1970) Mints et al. (1992) Rosenblum et al.(1990) Ramsay et al. (1989) Niu et al. (1992) Lieb et al. (1985) Clark et al. (1973) Gayler et al. (1955) 32 Salmonella Oranienburg watermelon Poona cantaloupes Saint-Paul beansprouts Stanley alfalfa sprouts Thompson root vegetables Dried seaweed CDC (1979) CDC (1991) Salmonella Salmonella O.Mahony et al. (1990) Mahon et al.(1997) Kano et al.(1996) Salmonella Salmonella 10 Salmonella Kano et al.(1996) Dunn et al. (1995) Shigella flexneri Mixed salad Shigella sonnei Iceberg lettuce Shigella sonnei Tossed salad Vibrio chlolerae Salad crops & vegetables Kapperud et al. (1995) Martin et al. (1986) Shuval, et al. (1989) Pathogens of most concern Salmonella The antigenic scheme for classifying salmonellae recognizes more than 2300 serovars and, 20 while all can be considered human pathogens, only about 200 are associated with human illness.

Shigella Bacillary dysentery or shigellosis is caused by Shigella, of which there are four species: 25 S.dysenteriae, S. flexneri, S. boydii and S. sonnei (Maurelli and Lampel, 1997). Most cases of shigellosis result from the ingestion of food or water contaminated with human faeces. Like salmonellae and other pathogens present in faeces, Shigella can contaminate raw fruits and vegetables by several routes, including insects and the hands of persons who handle the produce, although shigellosis is more often transmitted from person to person.

Escherichia coli Escherichia coli is common in the normal microflora of the intestinal tracts of humans and other warm-blooded animals. Strains that cause diarrhoeal illness are categorized into groups on the basis of virulence properties, mechanisms of pathogenicity, clinical syndromes and antigenic 35 characteristics. The major groups are designated as enterotoxigenic, enterohaemorrhagic, enteropathogenic, enteroinvasive, diffuse-adhering and enteroaggregative (Doyle et al., 1997).

Campylobacter Campylobacter jejuni is a leading cause of bacterial enteritis in many countries. Reservoirs of 40 this pathogen include several wild animals as well as poultry, cows, pigs and domestic pets (Nachamkin, 1997). While consumption of food of animal origin, particularly poultry, is largely responsible for infection, Campylobacter enteritis has also been associated with the consumption of raw fruits and vegetables (Bean and Griffin, 1990; Harris et al., 1996). Although 33 Campylobacter does not grow at temperatures below 30 °C and is sensitive to acid pH, it can survive on cut fruits for sufficient time to be a risk to the consumer (Castillo and Escartin, 1994).

Yersinia enterocolitica Yersinia enterocolitica can be found in a variety of terrestrial and freshwater ecosystems, 10 including soil, vegetation and water in lakes, rivers, wells and streams (Kapperud, 1991), but most isolates from these sources lack virulence for humans. Pigs, however, frequently carry serotypes capable of causing human disease. The ability of Y. enterocolitica to grow at refrigeration temperature and its documented presence on raw produce raises concern about the potential of salad vegetables as causative vehicles of yersiniosis in humans. Seven per cent 15 of carrot samples obtained from eating establishments in France were reported to contain serotypes of Yersinia that may be pathogenic to humans (Catteau et al., 1985). In another study (Darbas et al., 1985), 50% of raw vegetables analysed contained nonpathogenic strains of Yersinia. Incidence was higher on root and leafy vegetables than on tomatoes or cucumbers. Certainly, application of improperly composted pig manure to vegetable fields should be 20 avoided to reduce the possibility of pathogenic strains being present on produce when it reaches the consumer.

Listeria monocytogenes Listeria monocytogenes is present in the intestinal tract of many animals, including humans, so 25 it is not surprising that the organism can also be found in the faeces of these animals, on the land they occupy, in sewage, in soils to which raw sewage is applied and on plants which grow in these soils (Van Renterghem et al., 1991). The organism also exists in nature as a saprophyte, growing on decaying plant materials, so its presence on raw fruits and vegetables is not rare (Beuchat, 1992; 1996a; Beuchat et al, 1990). Surveys of fresh produce have 30 revealed its presence on cabbage, cucumbers, potatoes and radishes in the U.S.A (Heisick et al., 1989), ready-to-eat salads in the U.K. (Sizmur and Walker, 1988), the Netherlands (Beckers et al., 1989), N. Ireland (Harvey and Gilmour, 1993) and Canada (Odumeru et al., 1997), tomatoes and cucumbers in Pakistan (Vahidy, 1992), and bean sprouts, sliced cucumbers and leafy vegetables in Malaysia (Arumugaswamy et al., 1994).

Staphylococcus aureus Staphylococcus aureus is known to be carried in the nasal passages of healthy food handlers and has been detected on raw produce (Abdelnoor et al., 1983) and ready-to-eat vegetable salads (Houang et al., 1991). However, enterotoxigenic S. aureus does not compete well with 40 other microorganisms normally present on raw fruits and vegetables, so spoilage caused by nonpathogenic microflora would probably precede the development of the high populations of this pathogen that would be needed for production of staphylococcal enterotoxin.

Clostridium species 34 Spores of Clostridium botulinum and Clostridium perfringens can be found both in soil and on raw fruits and vegetables. The high rate of respiration of salad vegetables can create an anaerobic environment in film-wrapped packages, thus favouring the growth of C. botulinum and botulinal toxin production. Botulism has been linked to coleslaw prepared from packaged, shredded cabbage (Solomon et al., 1990) and chopped garlic in oil (St. Louis et al., 1988). 10 Studies have revealed that C. botulinum can produce toxin in polyvinyl film-packaged (Sugiyama and Yang, 1975) and vacuum-packaged mushrooms (Malizio and Johnson, 1991). It is important that the permeability characteristics of packaging films minimize the possibility of development of anaerobic conditions suitable for outgrowth of clostridial spores. Recognizing that anaerobic pockets may develop in tightly packed produce, even when films have high rates 15 of oxygen and carbon dioxide permeability, an additional measure to prevent growth of C. botulinum is to store produce at less than 3°C.

Bacillus cereus Spores of enterotoxigenic strains of Bacillus cereus are common in most types of soil. Some 20 strains can grow at refrigeration temperatures. Foods other than raw fruits and vegetables are generally linked to illness implicating B. cereus. Illness associated with eating contaminated soy, mustard and cress sprouts has, however, been documented (Portnoy et al., 1976). Human illness tends to be restricted to self-limiting diarrhoea (enterotoxin) or vomiting (emetic toxin). However, emetic toxin-producing strains have produced liver failure and death by the food-25 borne route.

Vibrio species Vibrio species are generally the predominant bacterial species in estuarine waters and are therefore associated with a great variety offish and seafoods. There are 12 human pathogenic 30 Vibrio species, of which Vibrio choierae, V. parahaemolyticus and V. vulnificus are of greatest concern (Oliver and Kaper, 1997). Vibrio choierae is the causative agent of cholera, one of the few food-borne diseases with epidemic and pandemic potential. Carriage of the organism by infected humans is important in transmission of disease. Water can become contaminated by raw sewage.

Viruses Viruses can be excreted in large numbers by infected individuals (Cliver, 1997). Although viruses will not grow in or on foods, raw fruits and vegetables may serve as vehicles for infection. 40 Many food-associated outbreaks of hepatitis A have been recorded (Cliver, 1997). In most instances, these outbreaks have not appeared to depend on the stability of the virus in the food.

Shellfish taken from waters contaminated with human faeces have been the vehicle in most outbreaks, but any food handled by an infected person may become contaminated and transmit infection (Cliver, 1985). Hepatitis A infection has been linked to the consumption of lettuce (Rosenblum et al., 1990), diced tomatoes (Williams et al., 1994), raspberries (Ramsay and Upton, 1989; Reid and Robinson, 1987) and strawberries (Centers for Disease Control and 10 Prevention, 1997a; Niu et al., 1992). Hernandez et al. (1997) suggested that lettuce contaminated with sewage could be a vehicle for hepatitis A virus and rotavirus. Lettuce obtained from farmer's markets were reported to contain hepatitis A virus. The extent to which hepatitis A and other viruses are removed from the surface of fruits and vegetables upon treatment with chemical disinfectants is not known.

The number of cases of food-borne disease caused by Norwalk-like viruses (i.e. Small Round Structured Viruses, or SRSV) appears to be on the increase (Bean and Griffin, 1990).

Outbreaks have a pattern of transmission resembling that of hepatitis A. Ice made from contaminated water has been implicated as the vehicle in more than one outbreak but salad 20 items have also been linked to Norwalk-like gastroenteritis (Karitsky et al., 1995). Workers who have prepared salads linked to viral gastroenteritis have been shown to have high antibody titers to Norwalk virus (Griffin et al., 1982; Gross et al., 1989; Iverson et al 1987). A non-typical outbreak of Norwalk virus gastroenteritis associated with exposure of celery to non-potable water has been reported (Warner, 1991). Studies have shown that viruses may persist for 25 weeks or even months on vegetable crops and in soils that have been irrigated or fertilized with sewage wastes (Larkin et al., 1978). Rotaviruses, astroviruses, enteroviruses (polioviruses, echoviruses and coxsackie viruses), parvoviruses, adenoviruses and coronaviruses have been reported to be transmitted by foods on occasion (Cliver, 1994). At least one echovirus outbreak has been attributed to contaminated raw shredded cabbage (New York Department of Health, 30 1989).

Chlorine Dioxide In The Dairy Shed Environment "The test chemical demonstrated effective bactericidal action, i.e. >log 5 reduction (or 99.999 3 5 kill, against all test organisms in 30 seconds of contact/exposure, except for bacillus cereus.

The exposure time required to obtain an effective log reduction of Bacillus cereus is in excess of 30 minutes."... Hills Laboratories test on the Southwell Product The test organisms were: 40 • Bacillus cereus • Campylobacter jejuni • Escherichia coli • Lactobacillus casei • Listeria monocytogenes 36 • Salmonella menston Applications: • General sanitiser • Biofilm removal • Spray for the treatment of mastitis.

Microbiological effectiveness of Chlorine dioxide at cold temperatures Microorganisms CI02 consumption Contact time Deactivation (ppm) (min) (%) Saccharomyces diastaticus (70 per cent sporulated) 1.3 ppm 10 99.999 Pichia (Hansenula) anomala (20 per cent sporulated) 3.8 ppm 5 99.999 Lactobacillus frigidus 2.5 ppm 5 99.999 Pediococcus damnosus 2.5 ppm 5 99.999 Enterobacter Cloacae 2.1 ppm 5 99.999 Damnosus and Enterobacter cloacae were used as test germs. The bacteria were in the lag Phase where they show an increased disinfectant tolerance due to lacking fissiparous scars. The used sporulating yeast forms are also particularly resistant to disinfectants. The experiments showed that Chlorine Dioxide is outstandingly appropriate for the killing of beverage relevant bacteria when the residence time amounts to five minutes. Even at 4 °C, a 40 complete killing of persistent sporulating yeasts can be expected after ten minutes at the latest. Therefore, this method is perfectly appropriate for disinfection purposes in the beverage industry even at temperatures of about 4°C.. 37 Experiments carried out by the same institute showed that other disinfectants with higher concentrations were by far not as effective as the Chlorine Dioxide Method.

A salicylic acid product with a 0.5 per cent concentration did not achieve a quantitative killing rate with the used microorganisms after 30 minutes of residence time. Even after 30 minutes, the beverage specific vermin remained significantly traceable, only enterobacter cloacae was quantitatively detected in this period.

Chlorine Dioxide - Applications - Poultry EXTENDER is used in food processing applications with a number of the following beneficial properties. 1. It does not have any pH limitations. 2. Its disinfectant (sterilisation) capabilities are not diminished at all in the presence of fats, oils, proteins, body fluids etc. because it has very selective and very few chemical reactions. 3. It is strongly soluble in water, therefore, it has a long-lasting residual which reduces the potential for cross infection or re-contamination. 4. It is a broad spectrum, fast acting disinfectant, effective against a wide range of bacteria, spores, fungi, and viruses at relatively low concentrations and short contact periods. 5. It is colourless, has a mild medicinal odour, low corrosivity to metals and the lowest acute toxicity rating from the EPA. 6. High efficacy against E.Coli, salmonella, listeria, aspergillus, penicillium, staphylococcus etc. 7. High efficacy is obtained irrespective of pH. 8. Non-corrosive and non-staining of equipment. 9. Easy to apply and to monitor.

. Meets HACCP (food safety) management requirements. 11. Cost effective.

Poultry Processing 38 Chlorine Dioxide has been used very successfully in poultry processing, as a processing aid that is added to process water maintaining good microbial quality thereby impacting on the quality maintenance and shelf-life of the produce.

Areas of Application The following would be the points of application 1. Scalding tanks 2. Carcass sprayer 3. Spin chiller 4. Inside outside carcass washer . Dip tanks for fallen birds Dosages 1. Treatment of the scalding tank water would be done at a dosage of 300ml per 1000 litres of water and the residual would thereafter be controlled at 10 -15 ppm. 2. Treatment of the carcass sprayer water would be at 500ml per 1000 litres of water and controlled at a dosage of 25 -50 ppm. 3. Treatment of the spin chillers would be done at a dosage of 300ml per 1000 litres of water thereafter maintain a 10 -15 ppm residual. A 25 - 50 ppm (at dosage of 500ml -1L per 1000 litres of water) dip solution could be made up for any birds that accidentally 25 fall on the floor.

FISHING TUNA LONG LINER IN TROPICAL MARINE WATERS.

Tuna is caught, gutted and suspended in refrigerated seawater at 0.5 deg. C. and stored for 30 between 3 to 9 days before landing and packaging.

Processing steps: Fish gutted Immersed in RSW hold 2 Litres of Clo2 per 1000 Litres RSW Advantages/benefits: Voyage time now sixteen days High oxidation power guarantees sufficient disinfection Appearance of gills and natural colour better than prior storage method Fish is considered to be of a higher quality 39 Better customer acceptance.

RAW SHRIMPS/PRAWNS Raw shrimp from farms (natural sea or rivers) are sent to a factory for processing.

Processing steps: Washing 5-10 ppm CI02 Sizing and peeling, washing with 2-3 ppm CI02 Final rinse 0.2 - 0.5 ppm CI02 15 Freezing Method of concentration control: Contact water meter, or by measurement Advantages/benefits: High oxidation power guarantees sufficient disinfection No influence by pH No smell or taste after final rinse water Better customer acceptance compared to chlorine treated shrimp 25 Improvement of TPC values MALODOROUS FISHING VESSEL The vessel was experiencing bad odour problems. It was suspected that a crack had appeared 30 in the hold wall that allowed organic material to pass into the foam insulation and generate bacteria causing the malodours and also resulted in the degradation of the fish because of unacceptable bacteria levels.

Processing steps: 5-10 ppm added to chilled seawater through a venturi and sprayed on the catch.

Chilling Method of concentration control By measurement 40 Advantages/benefits: High oxidation power guarantees sufficient disinfection No influence by pH No smell or taste after treatment 40 Better customer acceptance compared to chlorine treated fish Improvement of TPC values No malodours ICE PLANT A drum type ice making machine with the capacity of 2 MT per hour. The ice is used to pack fish in boxes.

Processing steps: 400 mis per tonne added to town water supply through a dosage pump.

Advantages/benefits: High oxidation power guarantees sufficient disinfection Dwell time of Clo2 release on contact with fish No smell or taste after treatment 20 Ice drums cleaned of bio-film allowing better contact with drum giving better ice Keeping qualities offish enhanced GENERAL SANITATION VEGETABLE CRATE WASHING PLANT Processing steps: Washed in detergent Passed through sanitation side and sprayed with CI02 10 ppm Advantages/benefits High oxidation power guarantees sufficient disinfection Cleaner appearance of crates No smell after treatment SANITATION FISHING HOLDS Processing steps: Gross filth removed and washed in detergent 40 Cleaned surface sprayed with 10 ppm CI02 solution Advantages/benefits High oxidation power guarantees sufficient disinfection E.Coli is below level of detection 41 No concerns about Listeria as CI02 will easily kill Listeria at low temperatures.

TPC at day zero is consistent and always less than 7 x 104 SOUTHWELL WATER TREATMENT OVERVIEW The Southwell system complies with the Drinking water standards of New Zealand and the product is listed as C61, Water treatment in Manual M15, New Zealand Food Safety Authority.

Potable water (Fit to drink) must comply with the Drinking water standards and as such have no coliforms present and comply with the listed criteria such as Iron and Manganese control.

OBJECTIVES To remove bacteria, cysts and undesirable metals from the water supply.

Bacteria and Cysts Take a review of the water supply and determine the level of contamination. The amount of chlorine dioxide required to decontaminate the water supply is proportionate to the degree of contamination.

Iron, Manganese etc.

Determine the quantity of the undesired substance and refer to the chart below.

SYSTEM DOSING Dosing the system depends entirely on the degree of sophistication of the application.

You could be faced a water supply sourced from ground water and once treated it will be consumed. In that case the determination of the degree of contamination, or even suspected degree of contamination, is to be measured or gauged and then the following formula should be 35 applied; Heavily contaminated 1 Litre 5000 Litres of water Mildly contaminated 1 10000 40 Low contamination 1 15000 In a system where tanks and pumps are available we can be more specific 42 If the flow of water can be measured, timing how quickly it fills a container of a specific volume would judge the amount issuing from a tap and accurate measurements can be made.

Assessments of the degree of contamination of the existing system must be made as to the amount of amassed bio-film with the system. The system includes; processing and storage 10 tanks and the interlinking pipes.

A shock dose introduced at 10 grammes of Product per tonne of storage will remove any algae or bio-film held in the system. In heavily contaminated systems debris will come through the tap. It is best to run the system until the water runs clear.

The next thing to determine is the presence of iron and/or manganese. In systems contaminated with these metals it is normal to have a tank that is used for their removal. The following table explains the system and the quantities for removal of various contaminants in the water supply. The product must be added to the outflow side of this tank before the water enters 20 a filter (if any) and is independent of any product introduced as a sanitizer as all the chlorine dioxide may be used up in removing the metals. The product passes through the filter and is ready to be dosed for sanitation.

Sanitation dosing is very simple. Dose at 1.5 grammes of product per tonne of water.

Flow of water per hour Amount of Chlorine Dioxide required (1000 Litres = 1 tonne) Centilitre 1000 1.5 5000 7.5 10000 15 50000 75 100000 150 To treat a flow rate of 165,000 Litres of water per hour add 100000+50000+10000+5000 = 165,000 150+75+15+7.5 = 197.5 centilitres per hour 40 CONTAMINENTS REMOVAL ALDEHYDES Aldehydes oxidize to the corresponding carboxylic acid. 43 Formaldehyde initially to formic acid and finally to carbon dioxide Paraformaldehyde can be depolymerised and eliminated completely by oxidation with chlorine dioxide.

AMINES AND MERCAPTANS Between pH 5 and 9, 4.5 parts by weight of chlorine dioxide instantaneously oxidises 1 part by 10 weight of a mercaptan (expressed as sulphur) to the respective sulphonic acid/sulphonate compound, destroying the mercaptan odour.

Similarly, chlorine dioxide reacts with organic sulphides and disulphides, destroying the original odour.

The oxidation of amines depends on the pH of the reaction mixture and the degree of 1 5 substitution of the amine.

Between pH 5 & 9, and average 10 parts by weight chlorine dioxide oxidises 1 part by weight of a tertiary aliphatic amine (expressed as nitrogen), destroying the amine odour.

At pH above 7, an average 5 parts by weight of chlorine dioxide oxidises 1 part by weight of a secondary aliphatic amine (expressed as nitrogen) removing all traces of amine odour.

The higher the pH of the reaction mixture (chlorine dioxide and tertiary and/or secondary aliphatic amines), the more rapidly oxidation proceeds.

AMMONIA PLANT Chlorine dioxide is chosen because of its non-reactivity with the ammonia commonly present in this system.

The starting Cl02 feed was 2 mg/l based on the total of system capacity and make up water over a 4-hour treatment period, once each day. Shortly after the initiation of CI02 feed, a residual of free and available chlorine (as CI02 via DPD method) was attained and reached a maximum of 0.9 mail before the CI02 feed was suspended for the day. After CI02 feeding was stopped for the day, there was a gradual drop in CI02 residual. It should be noted here that 30 chlorine residuals, under the previous gas chlorine programme, were seldom observed.

Total microbio counts under the previous chlorine program averaged approximately 15,000 organisms/ml. During the CI02 program, these counts have dropped to 1-5 organisms/ml and often sterile plates are observed.

CYANIDE DESTRUCTION Chlorine dioxide oxidises simple cyanide to cyanate (a less toxic substance) and/or carbon dioxide and nitrogen. The end products depend on reaction conditions. 44 In neutral and alkaline solutions below pH 10, and average 2.5 parts by weight of chlorine dioxide oxidises 1 part by weight of cyanide ion to cyanate. [1] Above pH 10 an average 5.5 parts by weight of chlorine dioxide oxidises 1 part by weight of cyanide ion to carbon dioxide and nitrogen. [3] Between pH 8 and 10 a mixture of by-products is produced [2] Chlorine dioxide does not react with cyanate ion, nor has it been observed to form cyanogen chloride during the oxidation of cyanide.

Chlorine dioxide also oxidises thiocyanate to sulphate and cyanate. In neutral solutions, an average 3.5 parts by weight of chlorine dioxide oxidises 1 part by weight of thiocyanate ion. [4] [1] pH in the range 7.0 to 8.0 CN" CI02 CNO" (Cyanate) [2] pH in the range 8.0 to 10.0 CN" CI02 mixture of products shown in 1 and 3 [3] pH greater than 10.0 2CN" CI02 2COz + N2 [4] pH 7 SCN" CI02 OCN" + S042" IRON Above pH 5 an average 1.2 parts by weight chlorine dioxide oxidises 1 part by weight soluble iron (ferrous) to insoluble iron (ferric).

CI02 + 5Fe(HC03)2 + 3H20 ——► 5Fe(OH)3 + 10C02 + H+ + 35 CI" Above pH 5 the resulting ferric iron is 99% removable by a 0.45 micron filter after 5 minutes. 45 MANGANESE The advantage chlorine dioxide has over chlorine is its speed of reaction. Chlorine reacts so slowly that manganese ions may still be in the water distribution system after 24 hours. Chlorine dioxide reacts much more rapidly with manganese oxidising it to manganese dioxide. After 5 minutes contact time, 99+% of the manganese may be removed through a 0.45 micron filter. 10 2.45 parts by weight of chlorine dioxide oxidises 1 part by weight of manganese. Best results are obtained when the pH is above 7. 2CI02 + 5Mn2++ 6H20 > 5Mn02+12H++ 2Cf NITROGEN COMPOUNDS Nitrogen oxides are hazardous and corrosive. Nitrous oxide (NO) and nitrogen dioxide (N02) 15 are industrial effluents which result from fuel combustion, nitric acid manufacture and use, and from metal finishing operations which use nitrates, nitrites or nitric acid. Other sources include chemical processes in which nitrogen compounds are used as reagents.

Chlorine dioxide has been used to scrub these contaminants. Nitric oxide contained in gas discharges from coke kilns may be eliminated by chlorine dioxide oxidation.

The process is particularly convenient for continuous operation.

PHENOL DESTRUCTION Surface water often contains phenols from industrial effluents. Undesirable phenolic wastes are produced in the chemical, plastics, coke and petroleum refining industries. If chlorine is used for oxidation, highly toxic chlorophenols are formed. These chlorophenols can also cause taste and 25 odour problems in drinking water. Orf/jo-chlorophenol is the most offensive of the phenol compounds. It is objectionable at concentrations as low as 1-2 ppb.

Treatment with chlorine dioxide can destroy chlorophenols. Below pH 10,1.5 parts by weight of chlorine dioxide oxidises 1 part by weight of phenol to benzoquinone [1]. Above pH 10 an average of 3.3 parts by weight of chlorine dioxide oxidises 1 part by weight of phenol to a 30 mixture thought to be low molecular weight, non-aromatic carboxylic acids (such as oxalic and maleic acids). At pH 7 the phenol reaction is rapid and complete; all phenols are consumed. pH in the range 7.0 to 8.0 C6H5OH + CI02 (Benzoquinone) pH in the range 8.0 to 10.0 C6H5OH + CI02 mixture of products shown in 1 and 3. 46 pH greater than 10.0 C6H5OH + CI02 HOOCCH = CHCOOH + HOOC - COOH (Maleic acid + Oxalic acid) SULPHIDES Many industrial processes and waste water/effluents produce sulphide containing gases and waste products. These gases are frequently scrubbed with alkaline solutions and require treatment before discharge.

Between pH 5 and 9, an average 2.5 - 5 parts by weight of chlorine dioxide instantaneously oxidises 1 part by weight of hydrogen sulphide (expressed as sulphide ion) to the sulphate ion.

S 2 + CI02 S042" THM PRECURSORS The key to understanding why chlorine dioxide is so effective can be found in the differences in the reactions of chlorine dioxide and chlorine with trihalomethane (THM) precursors such as humic and fulvic acids.

Chlorine reacts with THM precursors by oxidation and electrophylic substitution to yield both volatile and non-volatile chlorinated organic substances (THMs).

Chlorine dioxide however reacts with THM precursors primarily by oxidation to make them non-reactive or unavailable for THM formation. This means that pre-treatment with chlorine dioxide has an inhibiting effect on THM formation when chlorine is subsequently used.

Bactericidal, Cysticidal, Oocysticidai and Virucidal Effects of Chlorine Dioxide in Contaminated Water Chemical disinfection of drinking water is by far the most convenient approach to control transmission of infectious agents through water-borne route. However, problems include toxic 30 byproducts resulting from the use of disinfecting agents, and the ability of certain microorganisms to resist the inactivation, particularly protozoan cysts and oocysts.

According to the US EPA Guide Standard and Protocols for Testing Microbiological Water Purifier, for a microbiological water purifier to successfully pass the evaluation test, it must remove, kill or inactivate all types disease-causing microorganisms from the water, including 35 bacteria, viruses and protozoan oo(cysts) so as to render the processed water safe for drinking. Therefore, to qualify a microbiological water purifier it must inactivate all types of challenge microorganisms to meet the specified standards. Chlorine dioxide offers several advantages over chlorine for disinfection of drinking water. We have evaluated the ability of chlorine dioxide 47 to inactivate prototypic water-borne bacteria, protozoa and viruses. Experiments were conducted using EPA waters contaminated with bacteria (Klebsiella terrigena, ATCC 33257; Salmonella choleraesuis, ATCC 10708; Escherichia coli, ATCC 11229; Legionella pneumophila, ATCC 33153), viruses (Poliovirus type 1, ATCC VR-59; Rotavirus SA-11, ATCC VR-899) and protozoa (Cryptosporidium parvum oocysts from the USDA, Beltsville, MD, and Giardia muris 10 cysts from Oregon Health Sciences University) These experiments were conducted using the guidelines prescribed by the US EPA for testing microbiological water purifiers. Exposure to chlorine dioxide at a final concentration of 2 ppm in water for 10 minutes was effective in producing a > 6-log10 reduction in titer of all bacterial strains tested, at pH 5 + 0.2, 7 + 0.2 and 9 + 0.2 and at both 4 +10C and 20 + 50C, respectively. Similar treatment of rotavirus and 1 5 poliovirus produced >4 -Iog10 reduction in titer at neutral pH and pH 9.0.

The survival of bacteria and viruses were determined using standard assays.

Experiments are now underway to study the virucidal effect of chlorine dioxide at a lower pH. The protozoa part of the experiments included only spiked water at neutral pH which was 20 exposed to either 3 or 4 ppm of chlorine dioxide for 30-minutes. For determination of cystidal and oocystidal effectiveness of chlorine dioxide, a bioassay for used. Treatment of water with both concentrations of chlorine dioxide (3 and 4 ppm) totally abolished infectivity of both the cysts and oocysts for mice indicating > 3-log10. Chlorine dioxide has been found highly effective in inactivating those bacteria, protozoa and viruses that are common contaminants of drinking 25 water. In addition to the potential use of chlorine dioxide as water purifier as an alternative to chlorine, its applications in hospital settings, veterinary medicine and food industry will also be discussed.

The Use of Chlorine Dioxide in Disinfection of Wastewater Disinfection is the most important step in the preparation of wastewater for reuse in irrigation, industry, ground water recharge and in the long term for drinking water purposes. The hazards of reused wastewaters are primarily health risks of infection. Bacteria and viruses may damage the health of those who come in contact with wastewater unless it has been adequately treated. In some countries it is allowed to dispose of biologically treated effluents which contain 35 maximum geometrical average of 1000 total coliforms per 100 mL, or 200 fecal coli per 100 mL, during a period of 30 days. In California1 a level of 23 total coliform organism per 100 mL is required for irrigation of golf courses, parks and pastures grazed by milking animals. For direct irrigation of food crops, a level of 2.2 total coliforms per 100 mL is required.

WHO2 suggested health criteria for wastewater reused for irrigation of crops eaten raw not more 40 than 100 coliform organisms per 100 mL in 80% of samples. According to Kott3,4 20 to 40 mg/L of chlorine must be applied to biologically treated effluents for 6 hours to achieve a count of not more than 100 coliform per 100 mL. The Ministry of Health in Israel5 requires the disinfection of biologically treated effluents reused in irrigation, so that residual available chlorine should be 48 found after one hour contact time, in accordance with the type of plants irrigated. Table 1 summarizes the criteria for treated wastewater reused in irrigation in Israel. Usually it is not easy to achieve these effluents criteria by chlorine disinfection. Chlorine has some significant disadvantages when used for wastewaters disinfection due to its reactions with the organic constituents, forming chloro-organics and with ammonia, forming chloramines, which are less 10 effective than the free chlorine, and due to the large doses required to kill bacteria and inactivate viruses.

The purpose of this research is to investigate the feasibility of using CI02 as an alternative to Cl2 in the disinfection of effluents and ensuring environmentally acceptable finished water suitable for various reuses, resulting from a more efficient treatment. The research intends to study the 15 behavior of CI02 in wastewater effluents and in aqueous synthetic solutions containing organic and inorganic substances characteristic of effluents.

Experimental CI02 was studied on Haifa municipal sewage treatment plant effluents, from activated sludge and high rate trickling filters and on organic free media, such as distilled and tap water. Chlorine 20 dioxide was produced from sodium chlorite activated by HCI solution. Chlorine dioxide gas formed was driven off by bubbling air and carried through three empty traps in series before it was absorbed into distilled water, cooled with an ice bath. The CI02 mother solution was kept in refrigerator. Its concentration was determined at the beginning of each experiment.

The experiments were carried out using 3.5 L Duar glass flasks equipped with valve at the 25 bottom, a cover and magnetic stirrer. To 3.0 liters of effluents various doses of CI02 mother solution were added, and the solution was kept in darkness while mixing. Samples were taken at various times for chemical and bacteriological analyses. The samples for bacteriological tests were taken in sterile bottles, which contained 100 mg sodium thiosulfate to stop the disinfection activity by reduction of CI02 and HOCI. In spite of washing step the CI02 mother solution never 30 contained only CI02 but the following compounds as well: CI02", CI03", and free chlorine.

Analytical methods for the determination of Cl02 concentration in distilled water were studied, emphasizing the possibility of concentration determination of CI02 and other chlorine and oxygenated chlorine compounds after the contact with effluents. Knowing their concentration prior and after their addition to effluents is important for understanding the chemical reactions 35 taking place in this system.

The methods studied included amperometric titration, potentiometric titration or colorimetric end point determination. All these studied analytical methods are not simple and are time consuming.

The method chosen6"9 is an amperometric dead stop end titration, using PAO phenylarsine 40 oxide for determinations at pH 7.0 and above and Na2S203 for determinations at pH 2 to 3.0 and pH 0.1. Both titrations are based on measuring the amount of l2 liberated by oxidation of I" in Kl 49 by the various chlorine and oxygenated chlorine compounds at various pH levels. The analytical techniques were in accordance with procedures outlined in the following references: for N02~-N8, COD8, NH/-N10 and NOs'-N".

Results and Discussion The behavior of chlorine dioxide has been investigated on sewage treatment plant effluents and 10 compared to its behavior in organic free media, such as distilled and tap water. The aim of this research was to investigate whether secondary effects of chlorine dioxide application to effluents exist and if these affect its effectiveness as disinfectant. Particular attention has been paid to the interaction of CI02 with the inorganic and organic components of effluents and their effects on chlorine dioxide residuals. 1 5 The effects of varying chlorine dioxide doses, contact time and pH on residuals of CI02, CI02", CI03", HOCI and chloramines have been studied; as well as the effects on the destruction of total coliforms, fecal coli, streptococcus and coli phage, COD residual, organic nitrogen, ammonium, nitrite and nitrate ions.

Effect of Contact Time Table II summarizes the contact time effects on the various chlorine and oxygenated chlorine constituents and on the final pH, by addition of CI02 dose 7.84 mg/L as CI02, or 20.6 mg/L as Cl2. The concentration can be expressed as the specific constituent concentration or as Cl2. The range of contact times investigated was from 10 minutes to 24 hours. The mother solution consisted as follows: CI02 294.6 mg/L as CI02 (774.2 mg/L as Cl2) CI02" 201.4 mg/L as CI02" (423.3 mg/L as Cl2) Free Chlorine HOC1129.0 mg/L as Cl2 Table II shows that the effluents have an immediate CI02 demand, its concentration decreased to 3.0 mg/L as CI02 already after 10 minutes, 1.0 mg/L after 3.5 hrs and it had disappeared after 30 24 hrs. A part of the CI02 was converted into CI02", whose concentration increased with time. The effluents also have an immediate Ci02" demand. The CI02" ion concentration introduced with CI02 solution decreased initially from 5.4 mg/L as CI02" to 3.1 mg/L after 10 minutes, and subsequently increased with disappearance of CI02 up to a concentration of 7.0 mg/L after 24 hours. The residue CI02" increases by increasing the CI02 solution doses, but is always lower 35 than the initial concentration. It seems that the CI02" ion is most stable of the various oxygenated chlorine compounds in the effluents system under investigation. In this experiment the CI03" concentration was not determined. The free chlorine introduced with the CI02 mother solution is immediately consumed by the effluents, and it is not formed again. The free chlorine does not react with the ammonium ion in effluents, based on the absence of chloramines in the reacted 50 effluents. This was further verified in an experiment where the CI02 mother solution was added into a synthetic aqueous ammonia solution. The ammonium ion concentration did not change and chloramines were not found, although the system contained some free chlorine; thus in the present system chlorine does not react with ammonia in the presence of CI02. It seems that in effluents system the free chlorine reacts or oxidizes organic and inorganic substances and 10 disappears without forming chloramines. The total chlorine and oxygenated chlorine constituents expressed as Cl2 decreased with time. This behavior was found typical to effluents, tap water and distilled water systems: the residual concentration of CI02 decreases with time and disappears after several hours and sometimes at periods longer than 24 hours for higher doses. The effluents pH after adding the CI02 solution did not change during the first 60 1 5 minutes, and only then increased with time to a value of 8.5 at 24 hrs.

Investigation of the effect of contact time of CI02 with effluents on COD has shown as in Table III an immediate decrease of COD from 238 to 215 mg/L, the latter was constant up to 24 hrs. This COD reduction is caused by oxidation of one of the CI02 solution constituents and not by the bacteria, which were immediately killed. This Table also shows that neither CI02 nor free 20 chlorine has reacted with ammonia during 24 hrs. Presumably they reacted with other organic materials or oxidized other effluents' constituents, but did not react with the ammonia and did not form the chloramines. Moreover, the effluents' organic nitrogen did not decompose to ammonium ion, in the chlorine dioxide presence and the CI02 did not oxidized the ammonium ions to nitrites and nitrates, and did not form chloramines. This was evidenced by the constant 25 ammonium ion concentration (34.75 mg/L NH4+-N) and nitrates (0.08 mg/L N03"-N). This was further verified in a synthetic tap water system containing the following nitrogenous compounds: 15.2 mg/L NH4+-N, 8.8 mg/L N02"-N and 2.2 mg/L N03"-N to which 19.2 mg/L CI02 was added, at neutral pH as shown in Table IV. The CI02 immediately reacted with the nitrites, and disappeared after 10 minutes forming chlorite ions.

The CI02 demand for oxidation of nitrites was very high: 5.5 mg/L CI02 were required for each 1 mg/L N02"-N oxidized to nitrates. The 19.2 mg/L CI02 dose was not sufficient to oxidize all the 8.8 mg/L N02"-N due to nitrites presence in the synthetic tap water based solution, and their concentration decreased to a constant value of 5.3 mg/L. 0.25 meq/L of the nitrites were oxidized to nitrates, which concentration increased by 0.25 meq/L, from 2.2 mg/L N03"-N to a 35 constant value of 6.0 mg/L N03"-N.

The experiment was carried out at a neutral pH where CI02 accepts only a single electron, as follows: Cl02 + e CI02 The amount of CI02 (0.25 meq/L) disappeared according to this reaction agrees with the above 40 reported value of 0.25 meq/L N02"-N oxidized. Also, the CI02 specy did not react with the ammonia, and did not form chloramines in the synthetic systems and the ammonium ion 51 concentration remained constant, 45.2 mg/L NH4+-N, during the 24 hrs contact. The total nitrites and nitrates concentration remained constant 11 mg/L.

It is concluded that CI02 does not react with ammonia, but rapidly oxidizes nitrites to nitrates, in equivalent amount to its disappearance and chlorite ion formation. Moreover, the added chlorine and chlorite ion in the CI02 solution have not reacted with ammonium ion to form chloramines. 10 These experiments demonstrate that effluents from biological nitrification or nitrification- denitrification plants, which does not efficiently oxidize ammonia to nitrates, may contain high nitrites concentrations demanding high CI02 doses to oxidize nitrites to nitrates. Theoretically 4.8 mg CI02 are required to oxidize 1 mg N02-N and only then additional CI02 may be available for disinfection. Practically 5.5 mg CI02 were required to oxidize each 1 mg N02" -N. This is a 15 high disinfectant demand due to the presence of nitrites in nitrified effluents. An important conjecture is that since CI02 does not react with ammonia it is recommended as an efficient disinfectant for effluents from a conventional biological sewage treatment plant, without nitrification.

Effect of pH CI02 is well recognized as a strong disinfectant active in a wide pH range. In fact, its activity depends upon pH, which controls the number of electrons it accepts, and the resulting compounds formed.

At pH 7 and above CI02 accepts one electron as follows CI02 + e —> CI02 Since most of the reactions involved in water treatment take place within natural water and wastewaters pH range 7 to 8, a toxic chlorite ion is a major CI02 disinfection product. 60 to 70% of the CI02 is converted to CI02" after 24 hours. In an acidic pH range, CI02 is converted to chloride ion by accepting 5 electrons as follows: CI02 + 5e —> CI Similarly, at low pH free chlorine converts to CI" and CI02" is also converted to CI" by accepting 4 electrons as follows: CI02" + 4e --> CI" At highly acidic pH of less than 1.0 (pH 0.1) CI03" converts into CI" by: CIO3" + 6e —> CI" The pH effect was investigated in the pH range 3 to 10 by adding two doses of CI02 19.2 mg/L and 37.5 mg/L to effluents at a constant 20 minutes contact time, as summarized in Table V. The CI02 mother solution contained 52 CI02 = 624.5 mg/L as CI02 CI02" = 130 mg/L as CI02" Cl2 = 137 mg/L as Cl2 The initial concentrations were calculated from the given dose of CI02 mother solution added to the effluent.

The Table shows that if original effluents, having a pH 7.5 are acidified, the CI02 concentration is almost constant. In the alkaline direction the CI02 concentration sharply decreases to 0.11 mg/L CI02 from 19.2 mg/L and to 5.23 mg/L from 37.5 mg/L. Thus CI02 is reduced to CI02" or CI03" in alkaline solution. These ions are not considered as disinfectants.

On the acidic side CI02 is stable up to pH 4.0 and therefore may serve as an efficient 1 5 disinfectant within the pH range 7.5 to 4.0. At pH values lower than 3.5 its concentration is expected to decrease by reduction to CI" ion. Additionally Table V shows that free chlorine, at a dose of 19.2 mg/L has disappeared within all the pH range studied. Whereas, at the higher, 37.5 mg/L, dose, a residue was found only in the acidic pH. and it seems that the free chlorine rapidly reacts with organic compounds and disappears. It does not react with ammonia and 20 does not form chloramines. These results lead to a conclusion which contrasts literature reports stating that CI02 is more active in the alkaline range. It is concluded from the present study that due to its stability CI02 should be used as disinfectant at the acidic pH range. An additional advantage characteristics to this acidic pH range is that less chlorite ion, considered as a toxic material, inefficient as disinfectant, is formed.

Effect of Disinfection on Microorganisms Contact Time Effect The effect of CI02 on microorganisms was studied using biologically treated effluents from Haifa municipal sewage treatment plant. The effects of CI02 dose level, contact time and pH on killing total coliforms, fecal coli, streptococcus, total count and E-coli phage were studied. In addition 30 the CI02, oxygenated chlorine substances, free and combined chlorine residues were determined. The composition of CI02 mother solution in these experiments included: CI02 = 441.9 mg/L as CI02 CI02" = 23.0 mg/L as CI02" HOCI = 39.0 mg/L as Cl2 Residual concentrations of CI02, CI02, HOCI and chloramines in disinfection experiments using various doses of CI02 and contact times between 5 to 35 min. are summarized in Table VI and Table VII shows the survival of microorganisms in this experiment.

Effluents disinfection with CI02 has shown 98.9% kill of fecal coli after 30 min. contact time, using a dose of 2.7 mg/L CI02. This small dose is insufficient to efficiently kill after 5 min. 40 contact time. The killing efficiency was improved and contact times became shorter by 53 increasing the CI02 dose levels. A dose of 10.8 mg/L CI02 was sufficient to reduce the fecal coli from 3.3 x 10 in effluents to 14 within 20 minutes and two organisms survived after 30 min. contact time. Such CI02 dose bacteriologically qualifies the effluents for unrestricted irrigation, conforming to the specification criteria in Israel, WHO and also the strict California requirements. It is important to point out that for unrestricted irrigation chemical parameters of 10 effluents quality should also be accounted.

Effect of pH on Disinfection Effect of final pH on disinfection of effluents after 30 min. contact time, on total count, coliforms, fecal coli and E-coli phage (all MPNS expressed per 100 mL) is shown in Table VIII. The control initial concentrations are also given. 1 5 Disinfection efficiency of CI02 was tested at pH range between 4 and 10.0. The original pH of the effluents was 7.5. Initially samples were taken to determine only the pH effect on the microorganisms level in comparison with their original concentration and reported in this table as "control". Changes within the pH range studied were noted and subsequently accounted for. This Table shows that the pH is playing an important role on killing of these microorganisms. A 20 dose of 9.86 mg/L CI02 is efficient for killing in the acidic and neutral pH range up to pH 8.2, while in the alkaline range an increase of microorganisms survival can be noticed.

In conclusion this experiment has proven the high efficiency of CI02 in effluent disinfection achieving high bacteriological quality of the treated water. An efficient disinfection of the effluents is achieved with relatively low CI02 doses and short contact times at the neutral and 25 acidic pH range.

Conclusion In conclusion our preliminary secret trials indicate a surprising and unique aspect illustrating the potential of stabilized CI02 as an efficient disinfectant in effluents, as compared to standard CI02 systems. Our trials have shown that although a comprehensive understanding of the 30 mechanics of disinfection processes utilizing stabilised CI02 in effluent systems is still lacking the surprising and real advantages gained by using the stabilized CI02 methodologies of the present invention is of significant commercial value.

These studies are particularly important for a reliable disinfection of effluents intended for reuse. 35 Table I. Criteria for Wastewater Re-use in Irrigation in Israel type of irrigation coliforms per 100 ml residual available chlorine, contact time 1 hr cooked vegetables <250 0.15 mg/l 54 decidous fruits (80% of samples) football fields & golf courses unrestricted crops < 12(80%) parks & lawns < 3 (50%) 0.5 mg/l Table II. Effect of Chlorine Dioxide Contact Time on CI02, CI02", CI03" and HOCI Residuals in Effluents Treatment. CI02 Dose 7.84 mg/L.

Contact PH CI02 cio2- HOCI NH2CI Sum CI Time mg/L mg/L mg/L mg/L mg/L as as as as as as as CI02 Cl2 CIO/ Cl2 Cl2 Cl2 Cl2 0 min 7.75 7.84 .6 .4 11.30 3.44 0 .34 min 7.55 3.0 8.0 3.1 6.43 0.118 0 14.55 min 7.55 2.6 6.8 .0 .45 0 0 17.25 60 min 7.65 2.1 .5 .8 12.13 0 0 17.63 1,5 hr 7.75 1.9 .0 6.2 12.96 0 0 17.96 2,5 hr 7.90 1.5 3.8 6.23 13.10 0 0 16.90 3,5 hr 8.05 1.0 2.7 6.33 13.30 0 0 16.00 24 hr 8,50 0 0 7.0 14.66 0 0 14.66 Calculated Table III. Effect of Chlorine Dioxide Contact Time on Effluent's Ammonium Nitrites, Nitrates and COD. CI02 Dose 5.2 mg/L Contact Time pH ci02 ci02- hoci mg/L Sum ci cod nh4-n no2-n nos-n mg/L as ci02 mg/L as cio2- mg/L as cl2 mg/L as cl2 mg/L o2 mg/L mg/L mg/L Raw Effluent 0 7.4 — — — — 238 38.1 0.0 0.08 24 hrs 8.2 190 38.79 0.0 0.09 Effluent 0 .2 2.6 2.2 21.2 235 34.6 0.0 0.07 55 Calculated* min 7.3 1.9 2.8 0 .5 216 34.75 0.0 0.08 min 7.4 1.4 3.7 0 11.5 214 34.75 0.0 0.09 1 hr 7.4 1.0 4.2 0 11.6 214 0.0 0.15 2 hrs 7.5 0.8 4.6 0 11.7 216 0.0 0.09 3 1/2 hrs 7.6 0.4 .0 0 11.5 216 .54 0.0 0.09 24 hrs 7.95 0 .3 0 11.2 220 34.75 0.0 0.09 Concentration calculated due to dilution by adding 36 ml CI02 stock solution to 3.50 L effluents. Chloramines concentration is zero.

Table IV. Effect of Chlorine Dioxide Contact Time on Ammonium Ion, Nitrites and Nitrates Added to Tap Water, at Neutral pH. CI02 Dose 19.2 mg/L.

Contact Time pH CI02 CI02- HOCI + NH2CI Sum CI NH/-N N02--N N03"-N Sum N02" + N03" mg/L as CI02 mg/L as CI02" mg/L as Cl2 mg/L as Cl2 mg/L mg/L mg/L mg/L N 0 7.3 19.2 11.8 7.3 82.7 45.2 8.8 2.2 11.0 min 7.2 0 — 0.26 45.2 .3 .9 11.2 min 7.25 0 28.7 0.19 60.5 44.3 .3 6.0 11.3 1 hr 7.6 0.07 28.5 0.19 60.4 45.2 .3 6-1 11.4 3 hrs 7.9 0.03 29.5 0.19 62.2 45.2 .3 6.0 11.4 24 hrs 8.3 0.08 .5 0.20 64.5 44.1 .3 — Calculated after addition of stock solution.

Table V. Effect of pH on Chlorine Dioxide, Chlorite Ion and Free Chlorine Residuals in Effluents. CI02 Doses 19.2 mg/L and 37.5 mg/L. Contact Time 20 Minutes.

DOSE 19.2 mg/L as CI02 DOSE 37.5 mg/L as CI02 pH CI02 HOCI CI02.

Sum of CI pH CI02 HOCI CI02.

Ini* Final mg/L as CI02 mg/L as Cl2 mg/L as CI02. mg/L as Cl2 Ini* Final mg/L as CI02 mg/L as Cl2 mg/L as CI02. 7.5* 19.20* 4.2* 4.02* 63.0* 7.5* 37.50* 8.23* 7.8* 4 3.4 4.66 0 3.03 18.63 4.0 3.5 .84 0.55 56 4.6 4.37 0 .8.2 21.09 .0 4.95 26.88 0.18 6 6.45 4.95 0 .68 24.95 6.0 6.05 24.20 0.21 7.5 7.3 3.84 0 6.40 23.56 7.5 7.3 .27 0.86 9 8.8 1.27 0 .63 .70 9 8.8 .13 0 9.9 0.11 0 12.13 .81 9.8 .23 0 Ini* - Initial *The initial concentrations calculated from the given dose of CI02 mother solution. Chloramines concentration is zero.

Table VI. Residual CI02, CI02- & HOCI in mg/L in Disinfection of Trickling Filters Effluents with Various Doses of CI02. 2.7, 7.8 and 10.8 mg/L.

DOSE 2.7 mg/L as CIO 2 DOSE 7.8 mg/L as CI02 DOSE 10.8 mg/L as CI02 Contact time min.

CI02 as CI02 CI02. as C102.

HoCI as Cl2 Sum of *CI as Cl2 CI02 as CI02 CI02. as CI02.

HoCI as CI2 Sum of *CI as Cl2 CI02 as CI02 CI02. as CI02.

HoCI as Cl2 Sum of *CI as Cl2 0 2.7 0.14 0.24 3.08 7.80 0.41 0.69 8.90 .80 0.56 0.95 12.31 0 0.17 0 0.36 1.28 2.78 0 9.20 3.10 2.50 0 13.41 0 0.20 0 0.45 1.03 2.76 0 8.52 2.17 2.89 0 11.78 0 0.0 0 0 0.84 2.52 0 7.52 2.07 3.42 0 12.64 • Sum of CI = CI02 + CI02- + C 2 + mono chloramine as Cl2 Table VII. Disinfection of Trickling Filters Effluents with Various Doses of CI02 and Contact Times CI02 Dose mg/L Contact Time min.

Total Count per 100 mL Total Coliform per 100 mL Confirmed Coliform per 100 mL Fecal Coli EC Media per 100 mL Streptococcus per 100 mL 0 0 2x108 3.3 x107 3.3 x 107 3.3 x107 1.85x108 2.7 6.75 x107 2.4 x107 1.3 x 107 2.4 x107 8.4 x107 2.7 .3 x107 1.3 x107 2.4 x107 1.3x 107 2.4x10' 2.7 3.1 x 107 2.4 x106 2.4 x 106 3.5 x 10b .0 2.0x10® 2.4x10® 2.4 x 106 2.4 x106 .0 1.6 x 105 7.9 x103 1.1 x 102 7.9 x 10 .0 45 9.0 x 104 2.4 x 10a 7.9x10 4.6x10 .8 1.2 x10s 1.3 x103 1.3 x103 9.2 x102 2.7 x10s .8 2.9 x104 3.3 x 102 14 14 8.0 x103 .8 2.5 x104 7.9x10 7.8 2 ■ Table VIII. Effect of pH on Disinfection of Effluents with Constant CI02 Dose 9.86 mg/L and 30 Minutes Contact Time. MPN per 100 ml. pH Initial Final Total Coliforms Confirmed Coliforms Fecal Coli Total Count E-Coli Phage Control 7.5 — 4.9 x107 4.9 x107 4.9 x 107 2.4 x 10® 2.4 x 105 Control 4.6 ... 4.9 x107 3.3 x107 1.1 x107 ; 2.0 x 104 4.3 x104 Control .0 ... 1.3 x 107 1.3 x 107 1.3 x 107 4.0 x 104 2.4 x105 4.6 4.4 4.5 2 2 0 .5 .6 -4 OO 2 2 0 6.5 6.7 7.8 4.5 0 0 7.5 7.6 49 17 1.8 CM 0 1 X I 0 8.2 8.1 4.6 x 102 33 23 2 x 102 2 9.1 9.2 .4 x103 7.9 x102 2.7 x102 2.5 x 103 79 .0 .0 2.4 x104 1.3 x 104 1.3xio4 ; 6.8 x 102 79 EXAMPLE 1. The Southwell Dairy Treatment system This system is unique as to the manner in which the constituent parts are used and also how the ingredients making the chlorine dioxide diluent are assembled.

This study was undertaken to determine the efficacy of Southwell Extender, a proprietary oxy-chlorine sanitiser and a commercially available acid and alkali product in conjunction with a 58 reduced hot water regime and find if results regarding milk quality were compromised by the use of the system.

Costs of traditional chemicals were compared with use of the Southwell system. CONCLUSIONS Milk quality results were not compromised by using the Southwell system.

Actual power consumption readings showed a fall in the amount of power used.

November TOTAL 1852,47 January TOTAL 1075.26 May TOTAL 912.64 Cleaning material costs Alkali 140.00 Acid 800.00 Extender 1100.00 TOTAL CLEANING MATERIAL COSTS 2040.00 MATERIALS AND PROCEDURES The farm is located in the Northern Wairarapa and milks two hundred and sixty cows in a twenty 30 four a side herringbone shed.

The selected santiser, bio-film remover, was Southwell Extender, Chlorine dioxide in aqueous diluent < 1000 ppm (approval number h 2166a.) and it was obtained from Southwell Products Ltd..

It was noted that approval for the use of this product will be subject to the following conditions 35 as per NZFSA requirements: 1. To be used as part of a cleaning regime that includes hot water cleaning FIL Impact Blue, a caustic cleaner based on sodium hydroxide was obtained from a farm supplier and was used as the alkali cleaner during the period of the study FIL JetSet, a phosphoric acid, was the selected acidic cleaner. 40 All materials were used as recommended by the respective manufacturers i.e.

Extender 240 mis per 360 litres of water 59 The plant was cleaned prior to the beginning of the season with seven hot acid washes in the morning and with seven hot alkali washes in the afternoon. The plant was rinsed with potable water after each wash.

Southwell Products recommended the following regime....

Day 1 2 3 4 6 7 a.m.

Alkali Hot X * X * X * X Acid Hot * X * X * X _* p.m.

Extender cold X X X X X X X Followed by cold potable water rinse with a typical recycle time of ten minutes On the trial farm the following system was adopted Day 1 a.m.

Alkali Hot X Acid Hot p.m.

Extender cold -* Followed by cold potable water rinse with a typical recycle time of ten minutes As part of a secret trial the following system was a< Day 12 3 4 a.m.

Alkali Hot X Acid Hot p.m.

Extender cold -* -* -* -* -* -* Extender wash 40 Followed by cold potable water rinse with a typical recycle time of ten minutes All pipes and joins were cleaned four times in the season; at the start of the season, after calving, after mating and in March.

The vat was cleaned using two cold acid washes per week and the remainder using Southwell Extender. 45 Monitoring of the performance of the operation was done by Fonterra and energy consumption data was supplied by Genesis Energy.

RESULTS The milk quality results were as follows 2 3 4 5 6 7 ****** * * * * * lopted X 60 40 45 50 55 November SCC Bacto. Coliforms Inhabs Thermos Day 203 - 9 221 - 8 227 - 7 210 - 6 165 - 158 - 4 177 A+ December SCC Bacto. Coliforms Inhabs Thermos Day 173 - 9 169 A+ - 8 140 - 7 153 - 6 154 - 163 - 4 138 January SCC Bacto. Coliforms Inhabs Thermos Day 163 - 9 160 A+ 8 133 - 7 146 - 6 170 - 135 - 4 135 - February SCC Bacto. Coliforms Inhabs Thermos Day 189 - 9 231 - 8 238 - 7 165 A+ - - - 6 159 - 163 - 4 193 - March SCC Bacto. Coliforms Inhabs Thermos* Day 707 - - - 100 9 357 - - - 1600 81 162 - - - 1700 7 175 - - 1800 6 169 - - - 1200 191 - - - 1800 61 40 45 50 55 4 171 A+ 81 change to once a day milking 700 *Thermos due to perished rubber ware, dirty milk air lines and not attributable to plant cleaning April Day SCC Bacto.

Coliforms Inhabs Thermos 9 8 7 6 4 249 -240 - Unavailable Unavailable 253 A+ Power Consumption Actual Power reading at:- November (Contains part of October) Unavailable Business Night Business Day Daily fixed charge TOTAL 5455 Units used 2903 31 days at Cost Cents Extension 12,10 351.26 26.98 1471.76 95.00 29.45 1852.47 January Business Night Business Day Daily fixed charge TOTAL Units used 871 Cost Extension Cents 12.10 105.39 3468 26.98 935.67 36 days at 95.00 34.20 1075.26 May Business Night Business Day Daily fixed charge Units used 1094 Cost Extension Cents 12.10 132.37 2781 26.98 750.32 31 days at 95.00 29.95 TOTAL 912.64 62 Use in chilled and refrigerated water or brine to extend the shelf life of fish Previous to the introduction of the Southwell System high cost fish such as tuna caught in long line fishing voyages were gutted and wrapped in muslin and suspended by the tail in a tank containing chilled/refrigerated sea-water. This procedure was employed to retard the proliferation of spoilage mechanisms. Under the above regime voyage times were nine days.

The introduction of Southwell Extender chlorine dioxide in aqueous diluent has extended voyage times to sixteen days with any visible deterioration of the fish.

Trials on various fruits, vegetables and other products subject to rapid spoilage have shown considerable resistance to spoilage mechanisms.

Also surprisingly, this process has particularly use in the field of embalming and especially the 15 mortuary environment where a cadaver is flushed using chlorine dioxide in aqueous solution in conjunction with a use system devised by Mr. Adrian Featherstone of Mortech Industries (NZ) Ltd.

Use of chlorine dioxide with various additives to exhibit new uses Chlorine dioxide does not mix readily with other materials because of its oxidative effect. 20 However there are uses where it is desirable to have the aqueous diluent to be part of new carrier.

To this extend we have adopted a system of not trying to blend the diluent with the new material but rather use it as part of the reaction thereby extending its stability from "mix on the day" to periods in excess of three months.

Preferably, with a carrier such as glycerin to act as a fixative in the manufacture of a teat spray to be used in the dairy industry Preferably, with a surfactant to be used as a detergent for the lifting of fat and protein spoils while also having a disinfectant effect.

In one embodiment the stabilised chlorine dioxide solution is added to refrigerated sea-water 30 preferably at zero point five degrees Celsius (0.5 deg. C) at a rate of one litre (1 L.) per one thousand litres (1000 L.) of sea-water. The effect of the stabilised chlorine dioxide solution is to supress the growth of spoilage bacteria thereby allowing voyage times to be extended from nine (9) to sixteen (16) days.

This embodiment has been refined to add four hundred millilitres (400 ml.) of stabilised chlorine 35 to one thousand litres (1000 L) of water used in the making of ice in commercial ice making machines. When the ice is packed around fish the change in temperature releases the stabilised chlorine dioxide and has the supressing effect on spoilage bacteria as above 63 A further embodiment see stabilised chlorine dioxide introduced into cadavers with the effect that on contact with spoilage bacteria in the bodies system retardation takes place thereby holding back the natural decomposition of the body.

In agriculture and horticulture stabilised chlorine dioxide has been used as both a topical spray and also inoculated into the plant itself. introduction to biofilms Many bacteria are planktonic, that is they float around in water. Most microbiological work is done using these suspended cultures on water samples.

Most of the bacteria that cause problems are sessile, attached to a surface. Once bacteria attach to a surface they change.

The most obvious change is that they begin to excrete a slimy material, hence the source of the 20 derivation of the word jbiofilm. However, research is showing that biofilm is not merely the provision of the excretion of slimy material but rather they are showing that bacteria which attach to a surface turns on a whole different set of genes which effectively makes it a significantly different organism to deal with compared to the planktonic material.

Bacteria living in a biofilm do a number of things differently from the single planktonic cells of the same type of bacteria e.g. Pseudomonas aeruginosa, and these are: There is a division of labour in a biofilm where some cells utilise the available nutrients to turn on metabolic pathways. Other cells utilise degradation products (suspended solids, corrosion 30 products, dead bacteria and algal cells) to produce new cells that are dispersed into the biofilm environment.

In biofilms, bacteria (film forming fungi can also form biofilms) employ cell-cell communication which is now termed quorum sensing where they sense the level of increased cell population 35 density and they release and detect hormone-like molecules that accumulate in the surrounding aquatic environment as the bacterial cell density increases.

The biofilm having achieved this quorum sensing shows vast differences in heterogeneity from the same bacterial species in different environments. 40 The biofilm having achieved this quorum sensing status can begin to excrete toxins and polysaccharides, change the properties of the original bacterial cell, and change the shape of the biofilm. 64 CHARACTERISTICS OF BIOFILMS Biofilms consist of: water (85% to 95% by weight) Microbial cells Extra-cellular polymeric substances (EPS) such as polysaccharides, proteins and other biopolymers, Suspended solids, Corrosion products, Algal material, Fungi & Protozoa.

The biofilms grow in micro-colonies embedded in the EPS structure which are interspersed with less dense regions containing highly permeable water channels. Counting of individual micro-15 organisms in a biofilm is not practical and in addition a number of species in the growing biofilm can not be cultured.

Research has shown that there is no difference in the rate of colonization across different types of supporting material (glass, stainless steel, rubber lining). The actual number of viable cells in 20 the biofilm will differ in terms of absolute number of colonies.

Biofilm structure is very dependent upon fluid velocity of the water, nutrient load, temperature, pH, electrostatic potential, biocide concentration and biocide contact time. Change a process parameter and the biofilm structure changes. Biofilms can grow across a vacuum.

There are four ways by which detachment of biofilm from a surface takes place, Erosion, small particles from the biofilm surface being detached into the bulk fluid Sloughing, large pieces of biofilm being detached Abrasion, detachment by collision of solids 30 Grazing, removal of biofilm due to its consumption by higher organisms such as protozoa These four different methods of detachment each exert a different response in counting microbiological colonies in bulk water samples and they exert different effects on disinfectant or biocide efficacy.

Detachment of biofilm can occur by increasing the flow rate of water to greater than 3-4 metres per second. Fluid shear forces cause erosion whilst high fluid velocities cause abrasion and sloughing. 40 Sloughing of biofilm is caused by disinfectants or biocides.

Detachment of biofilm is dominated by the electrostatic interaction in cell to cell attachment. Change in electrostatic potential can change the biofilm structure. 65 The structure of biofilms is a function of the spatial distribution and homogeneity of the biofilm in a water circuit, hence, the importance of measuring spatial distribution of biofilm.

The structure of biofilms depend on the following,Turbulent flow produces homogeneous and slimy biofilms. Laminar flow produces a scattered biofilm with significant protuberances. 10 Laminar flow biofilms are more easily inactivated than turbulent flow biofilms.

Turbulent flow biofilms are more active as seen by the increase in respiratory conditions for the micro-organisms, have less EPS but higher protein content. (Proteins which contain glycine, lysine and histidine react with many disinfectants/biocides like chlorine, bromine, ozone, 1 5 glutaraldehyde, QAC's, peracetic acid products, hydrogen peroxide. Please note there is no reaction with chlorine dioxide) The effect of disinfectants or biocides is related to the age of the biofilm. Younger biofilms are easier to remove but age is relative for each system as age varies from minutes to days.

Shock dosing of a disinfectant or biocide has been demonstrated to be significantly more superior to continuous low level dosing in the removal or detachment of biofilms. In many cases the level of detachment of biofilm changes by factors of 10 to 100 times for shock dosing compared to continuous dosing.

The decrease in the susceptibility of biofilms to disinfectants or biocides has been proven to be influenced by phenotypic characteristics of the adherent cells and biofilm rather than biofilm structure, the various cells in the biofilm of the same bacterial type, that originally formed the biofilm undergo physical or chemical changes due to the formation of the biofilm thereby they 30 exhibit different properties to their planktonic relatives.

Biofilms do not grow in homogeneous structures. They change their shape, size and other chemical or physical characteristics across any given unit area and across the whole system, spatial distribution of the biofilm is a major factor in determining the ease of detachment of the 35 biofilm.

In potable water distribution systems biofilm formation leads to a deterioration of the microbiological quality of the treated water resulting in: Re-growth of coliforms of non-faecal origin 40 Multiplication of opportunistic pathogens like Aeromonas, Pseudomonas and Legionella Increased heterotrophic plate counts Colour, odour and taste problems Microbiologically induced corrosion (MIC) Induction of scaling 66 The provision of protective places for pathogenic bacteria Microbial measurement in potable water systems poses special problems mainly related to the low amount of bacteria present, low levels of nutrients in the potable water and their low activity.

The best suited techniques are those that are very sensitive to these small changes.

Impact of disinfectants/antimicrobials/biocides on biofilms Glutaraldehyde has been shown to provide a protective effect on cells against lysis and has no effect on biofilm at 200 ppm levels The most widely tested compounds used to control biofilm have been chlorine, hydrogen peroxide, Quaternary Ammonium and peracetic acids. These chemicals have been shown to have very poor to no effect on biofilm detachment.

Ozone has been shown to kill cells in the biofilm without any detachment of the biofilm. Re-growth of the micro-organism population 2 to 4 days later is evident with ozone treatment.

Biofilms have been shown to grow across UV lights quite readily.

The latest research by G. Gagnon, Dalhousie University in Canada has shown that chlorine dioxide and chloramines are very effective in the detachment of biofilms in potable water distribution systems There is no one mechanism rather researchers believe that there are 3 broad categories: Reduction of the antimicrobial concentration in the water surrounding the biofilm The antimicrobial agent is depleted to ineffectual levels before it gets to the biofilm.

Failure of the antimicrobial agent to penetrate the biofilm The antimicrobial agent is delivered to the surface of the biofilm but it does not effectively penetrate the biofilm. 40 Adoption of a resistant physiological (phenotype) by at least a fraction of the cells in the biofilm The antimicrobial agent permeates the biofilm but it is unable to kill micro-organisms because they exist in a phenotype state that confers reduced susceptibility. 67 The reduced susceptibility of biofilms has not been attributed to the usual mechanisms of mutation or acquisition of genetic elements that cause specific resistance genes that account for conventional antibiotic resistance. For these mechanisms to explain biofilm resistance, the genetic modification would have to appear in the biofilm but absent in the planktonic state, this 10 is not happening.

Some research has also shown that the amount of biofilm removed and the reduction in viable cell numbers in the biofilm were not correlated. Some antimicrobial agents cause significant killing but not much removal of biofilm and vice versa. This underscores the fact that biofilm 1 5 removal and cell killing are distinct processes and both need to be fulfilled to have a successful treatment.

Measurement showed that in an ice water system in one winery a residual of 1 ppm chlorine dioxide gives results while at another, good results were only obtained with 3 ppm residual.

Research has shown that a shock dose of an antimicrobial will do more damage to the biofilm than a low continuous dose and this is easily explained by the three mechanisms which explain antimicrobial resistance. There is a minimum inhibition concentration (MIC) that any antimicrobial requires before it can inactivate a bacteria cell.

It is obvious that the MIC for the same type of bacteria can differ from site to site which explains why one begins to get a good result but one week the bacteria counts are high again. A shock dose at this point will get on top of the problem.

Chlorine dioxide is a more effective antimicrobial than most other chemicals because of its small molecule; it is non ionic, it is a gas, it is highly soluble in organics, it does not react with polysaccharides, has very few chemical reactions and is stable in water with a measurable residual.

Even with these characteristics there is no "standard" level for removal of biofilm.

Overview To Biofilm Monitoring Bio-fouling is a biofilm problem it is an undesired deposition and growth of micro-organisms on surfaces such as heat exchangers, water storage and distribution systems and in medical applications. These biofilms cause significant economic losses. Any strategy which incorporates 40 anti-fouling technologies will be more cost effective if the extent of the biofilm could be monitored on-line in real time without destroying the biomass formation.

Current bio-fouling monitoring techniques rely on the removal of biomass from the system in the form of coupons that have been exposed to the fluid for a given period of time. These samples 68 are then analysed which is time consuming and requiring skilled personnel. Furthermore, current biofilm control technologies are based on Monitoring the process performance or product quality, the biofilm is detected only after it has already caused economic losses.

Biofilm monitoring is based on decisions made from the results obtained from bulk water samples. It has been shown above that there is no correlation or relationship between planktonic bacteria and sessile bacteria of the same type.

Biofilm is usually treated as a disease of the plant process water. If the organisms in the bulk water are killed a cure of the disease is made.

Disinfectants are used to kill the organisms in the bulk water, however, they will leave dead biomass in the system that accumulates and promotes re-growth of the organisms by using the dead biomass as a nutrient source. (In many instances the real problem is the biomass of the biofilm).

Some oxidising disinfectants (like chlorine dioxide) cleave the bonds between the extra cellular polymeric substances (EPS) which are responsible for the attachment of the biomass. This detached biomass needs to be inactivated, by shock dosing, so as to stop the re-growth potential.

Biofilms are resistant to many disinfectants like chlorine, ozone, peracetic acid because they only cause cell deaths and re-growth of the biofilm is evident. In these instances a "saw tooth curve" of micro-organism levels is evident.

In most instances the amount of nutrients in a system is not limited. Oxidants like ozone can actually increase the amount of assimable organic carbon content thereby increasing the biomass quantity.

Biofilms are evident some time after formation. Research has shown that detachment of the 35 biofilm is dependent upon its age, the type of disinfectant or biocide used; its concentration and contact time available in the system.

The general mode of operation is for the significant over use of poorly selected disinfectants or biocides that result in economic or environmental concerns and costs. 40 Contemporary bio-fouling control strategies operate with information from water samples and blindly applying disinfectants or biocides because they kill these organisms in the planktonic state.

Bio-fouling monitors operate on four levels 69 Measurement of the kinetics of deposition of material and changes to the physical properties of the deposit These systems cannot detect the difference between micro-organisms (biotic) and abiotic deposit components like corrosion deposition, suspended solids, scale and non micro-10 organisms. Kinetics based systems work on a variety of parameters like light scattering; turbidity measurements; electrochemical changes in conductance; redox potential and heat transfer exchange resistance.

Systems which can distinguish micro-organisms (biotic) and abiotic deposits in a biofilm 1 5 These systems can measure the kinetics of deposition of biofilms and some measure the spatial distribution of biofilms. They can be used to correlate biofilm structure with absorbance for a given set of plant conditions. They can also be used to monitor disinfectant or biocide efficacy by changes in biofilm structure.

These systems use infrared sensors, fluorescence or microscopic observations.

Systems that provide detailed chemical and or physical composition of the biofilm.

They use sophisticated spectroscopy and microscopy analysis and currently are only suitable for biofilm research and not for use in industry.

Systems can discriminate between living and dead organisms within the biofilm surface.

To-date no such equipment exists.

Bio-fouling monitoring is direct, on-line, in-situ, continuous, non-destructive real time information 30 regarding biofilm in a specific system. Industrial process water or potable water is not a sterile system hence there is a level of biofilm in all systems which is inherently present without causing problems to that system.

The difficulty lies in determining the "base-line" for each system.

Bio-fouling monitoring is basically a means of monitoring physical, chemical parameter(s) it is not a means of quantifying biofilm function.

Currently there is no way of doing this. 40 Biofilms do not conform to any mathematical model; they vary in thickness, density and physical or chemical composition from point to point in any given biofilm in any given process water system. Bio-fouling monitoring is a means of measuring and comparing specific parameter(s) in biofilms in a specific process over a period of time. 70 Optimising the type of disinfectant or biocide to be used, cleaner applications that require more sophisticated monitoring strategies and different bio-fouling removal technologies are going to become the state of the art techniques to optimise disinfectant or biocide usage.

Biofilm Control Strategies Selection of the right disinfectant/biocide and the most cost effective shock dose timing regime The applied dosing of the appropriate disinfectant or biocide in a biofilm control strategy will need to satisfy the following conditions:- Low redox potential No hydrolysis or dissociation in water Few chemical reactions particularly with polysaccharides, proteins, enzymes and b-polymers High solubility and stability in hydrocarbons 20 Identification of biofilm formation, above the level of the baseline biofilm that no time is wasted in remedial action Changes in process conditions alter the rate of colonisation and biofilm characteristics. Bio-fouling monitoring needs to be sensitive to these changes.

Each system will have different biofilm characteristics even if the same bacteria type is the 25 responsible organism, e.g. slime formers, SRB's etc. Dosing patterns will vary.

Detachment of biomass, in most cases, is important without causing process or product contamination. Only killing of cells prevents re-growth. (Soak and disinfect process off-line will achieve these results provided the disinfectant can remove biofilm).

Shock dosing in terms of concentration and time between intervals will vary from system to system. The only method of effectively monitoring the cost effectiveness of this treatment is by using a bio-fouling monitor which can monitor disinfectant or biocide efficacy.

Biofilms contain areas of highly permeable water channels. Disinfectants or biocides efficacy requires a diffusion time for the product through these channels. Over a period of time more 35 biofilm is removed and the disinfectant biocide shock dosing pattern will be reduced.

Bio-fouling control is a sophisticated science with no standard method to treat similar systems. There is a need for product optimisation used in conjunction with a bio-fouling monitor prior to attaining the desired results but this process will be far more cost effective then blindly adding a 40 disinfectant or biocide in the hope of controlling biofilms.

A number of techniques will be needed to be used to achieve the most cost effective treatment programme. 71 The focus of our bio-fouling control strategy will be centred on a biomass SCOPE to give us online real time information about the start of biofilm formation. At this point the biofilm is at its weakest state. The SCOPE unit will give a digital signal at the outset of the biofilm formation which will then activate a chlorine dioxide shock dose. The duration of this shock dose will be determined by the SCOPE and through empirical results from the monitored process 10 parameters.

As is evident from all the research on biofilms there is no "standard" method of removal and killing of biofilm. Each system is to be evaluated individually and in terms of the customer's requirements taking into account: Process performance Product integrity Regulatory issues HACCP, Eurogap, Food Safety, ISO 14000 environmental discharge regulations, FDA, EPA and EU approvals 20 Cost effectiveness Microbiological efficacy To achieve these requirements we will make use of: Soak And Disinfect Procedures: Chlorine dioxide has been shown to be hugely effective in the removal of biofilm biomass through the use of a soak and disinfect process.

The water storage and distribution system is treated with 5 to 15ppm residual chlorine dioxide 30 solution which is then held for periods of 1 hour to 24 hours at this residual.

The nett result is the removal of the biofilm biomass which can create its own array of problems.

Bio-Dispersants The concept of Bio-dispersants is widely used in the treatment of cooling systems. We now have an on-line means of determining the most cost effective bio-dispersant. Bio-dispersants are used on a shock dose basis and the intervals between shock doses can be optimised for each system in order to maximise results with costs. 40 Shock Dosing Chlorine Dioxide Most industries are happy to undertake continuous dosing of chemicals, chlorine dioxide included, however shock dosing will in fact give more effective results. The Biomass detector will allow us to wean industry off continuous dosing and use shock dosing. Where there are significant cost benefits to be derived from using shock dosing. 72 SYNERGISTIC ANTIMICROBIAL, BIOCIDE COMBINATIONS There is no ideal biocide, so there is room in the fight against BIOFILM, BIOMASS to use combination products for the most effective results. These combinations will be determined, by the nature of the process and extent of the problem, Ozone and UV light in potable water for the quick kill and chlorine dioxide for the residual Chloramines together with chlorine dioxide The chloramines is used as the residual source and the chlorine dioxide to breakdown the biofilm and nitrifying bacteria QAC (QUAT) products in combination with chlorine dioxide, QAC products have great wetting ability.

Some biocides have longer half-life than chlorine dioxide in cooling water systems so combinations would provide cost effective solutions.

Furthermore, bio-dispersants programmes with chlorine dioxide could well reduce the need for 25 re-tubing condensers in power plant circuits.

Non-oxidising biocides in combination with chlorine dioxide (an oxidising biocide) will provide maximum insurance against organisms showing resistance to any biocide. This is equally important in cooling towers as well as in the cleaning of poultry houses particularly in the latter 30 case against the spread of the quick mutating avian flu virus which is wreaking havoc in the poultry industry in Asia.

Biofilm control strategies will need to have multiple levels of attack not blindly taking surface water samples and adding a biocide at a rate that the customer deems affordable.

We are able to show our customers that we have the capability to measure the problem and the solution.

Whilst the invention has been described with reference to specific embodiments, it will be 40 appreciated that various modifications and improvements could be made to these embodiments without departing from the scope of the invention as set out in this specification.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. The applicant makes no admission that any reference constitutes 73 prior art - they are merely assertations by their authors and the applicant reserves the right to contest the accuracy, pertinency and domain of the cited documents. None of the documents or references constitute an admission that they form part of the common general knowledge in NZ or in any other country.

Equivalents Clause.

The Invention may also broadly be said to consist in the parts, elements and features referred or indicated in the specification, individually or collectively, and any or all combinations of any of two or more parts, elements, members or features and where specific integers are mentioned 15 herein which have known equivalents such equivalents are deemed to be incorporated herein as if individually set forth.

Modifications and Variations The invention has been described with particular reference to certain embodiments thereof. It will be understood that various modifications can be made to the above-mentioned preferred embodiment(s) without departing from the ambit of the invention.

Variations can include the steps involved to obtain the desired stabilised end product and 25 scalability.

The skilled reader will also understand the concept of what is meant by purposive construction.

The examples and the particular proportions set forth are intended to be illustrative only and are 30 thus non-limiting.

Throughout the description and claims of the specification the word "comprise" or variations thereof are not intended to exclude other additives, components or steps.

Kit of Parts It will also be understood that where a product, method or process as herein described or claimed and that is sold incomplete, as individual components, or as a "Kit of Parts", that such exploitation will also fall within the ambit of the invention. 40 In a preferred embodiment the invention includes within its scope a kit of parts, the kit of parts providing for a stabilised solution of CI02 for use as a sanitiser comprising in separate containers or as separate mixable compartments within the same container: 74 (A) a chlorite salt and (B) a suitable acid and (C) an additional chlorite salt and wherein components (A), (B) & (C) are combined at steps and in amounts effective to provide for enhanced CI02 stability 75

Claims (6)

1. RECEIVED at IPONZ on 14 February 2012 WHAT l/WE CLAIM IS: 1 A method of preparing a stabilised chlorine dioxide solution by the following means: (i) Take 500 grams of 80% sodium chlorite and dissolve in water (ii) add water to a two hundred litre container and (iii) add the solution prepared at step (i); (iv) at a level of one hundred litres of water add 500 - 1000 mis of HCI at a concentration of 32%w/v (v) at a water level of one hundred and fifty litres add a further 25grams of sodium chlorite and add one litre 16% wIv solution of HCI to the water and (vi) fill to 200 Litres.
2. 2 A method of preparing a stabilised chlorine dioxide solution according to claim 1 wherein the final volume to be achieved ranges from 1 Litre to 5000 Litres and components (i), (iv) and (v) are in equivalent stoichiometric ratios as found in claim 1.
3. 3 A method for the control or suppression of infection in a non-human which comprises applying at a desired location the stabilised chlorine dioxide solution prepared by the method of claim 1 or claim 2.
4. 4 The method of claim 3, wherein the stabilised chlorine dioxide solution is applied by conventional means including spraying, flushing, dowsing, wiping, pouring, orwicking.
5. 5 The method of claim 3 or claim 4, wherein the stabilised chlorine dioxide solution is applied simultaneously or sequentially.
6. 6 The use of stabilised chlorine prepared by the method of claim 1 or claim 2 in the manufacture of a biocidal composition for the control or suppression of infection. PIPERS, Patent Attorneys for Tadeusz KROGULEC 76