GB2293878A - Monitoring biocide content of industrial waters - Google Patents

Abstract

Biocide concentration in industrial process water, e.g. cooling water in towers or aircon system water, is monitored by (a) assaying the biocide concentration in a sample using bioluminescent microorganisms and (b) assaying the level of living biomass in a sample. Microorganisms used in (a) may be a photobacteria whose reduction in luminescence is measured on addition of the water sample and assay in (b) is typically based on an ATP measurement using a luciferin/luciferase system. Kits for carrying out the steps (a) and (b) are also claimed. Measurements give the level of biocide in the water and also its overall bacteria level thus showing whether or not the bacteria are proliferating.

Description

COMBINED ASSAY The present invention relates to a method of monitoring biocide in an industrial water system, a method of operating an industrial water system in which the effectiveness of biocide is monitored using the method and a kit for monitoring the effectiveness of biocide in an industrial water system.

Biocides are used to treat water which is used in a wide variety of industrial applications. Thus for example in cooling water systems, such as industrial cooling towers, the water used is not sterile and biocides are used to prevent the accumulation of bacteria growing within the system. In air-conditioning systems the treatment of the water used in the system with biocides is important to prevent the build up of bacteria in the system. Similarly, water which is used in paper-making is treated in order to prevent the growth of bacteria in water which comes into contact with the paper-making equipment.

A wide variety of different agents have been used for the control of such bacteria. Chemical agents which are biocides and which have been used widely include isothiazolones, methylene bis(thiocyanate), quaternary ammonium compounds and agents which release chlorine or bromine.

In order to control efficiently the level of bacteria in such systems it is desirable to monitor the level of biocide using an assay which can be quickly and conveniently used on-site to ensure that biocide is present in the system and also to ensure that bacteria are not growing out of control. Likewise, where more than one biocide is used in combination, as is frequently the case, it would be desirable to be able to distinguish between the biocides present in a system since they may have different efficacy against different types of bacteria.

Prior methods which have been used to assay the level of biocide in industrial water systems have suffered from several disadvantages. Notably, such assays rely upon the use of chemical tests using for example dyes which measure the level of biocide chemical present in a sample. A difficulty with such tests is that they may be non-specific and may for example be effected by the presence of compounds related in structure to the biocide but which do not possess biocidal activity. Such tests are moreover often laboratory procedures which take considerable time to perform and cannot be used as a convenient diagnostic onsite tool. In addition, such tests are purely chemical; they do not measure the concentration of biocide in a system which is available to biologically react with bacteria.

In addition, assay for the level of biocide alone in a system can be inappropriate since it may be that bacteria, resistant to the particular biocide present in a system may nevertheless proliferate and cause a potential hazard.

The present invention seeks to address these problems by providing a method of monitoring a water system which combines a convenient assay for the concentration of biologically available biocide together with an assay for living biomass in an industrial water system.

The present invention provides a method of monitoring biocide in an industrial water system, which comprises: (a) assaying the concentration of biocide in a sample removed from the system by contacting the sample with bioluminescent microorganisms and determining from the reduction in the level of bioluminescence, the concentration of biocide; and (b) assaying the level of living biomass in a sample removed from the system.

The present invention further provides a method of operating an industrial water system in which the effectiveness of biocide is monitored in accordance with the invention and in which the concentration of biocide and level of living biomass are maintained within predetevmined limits by the addition of biocide.

The invention yet further provides a kit for monitoring biocide in an industrial water system which comprises: (a) means for assaying the concentration of biocide in a sample removed from the system by contacting the sample with bioluminescent microorganisms and determining from the reduction in the level of bioluminescence, the concentration of biocide; and (b) means for assaying the level of living biomass in a sample removed from the system.

The present invention thus combines two assay methods which are used together to provide an overall method of monitoring the effectiveness of biocide in an industrial water system.

The assay for the level of biocide is based upon the rate at which biocide from the water system reacts with bioluminescent microorganisms, such as bacteria thus reducing the intensity of light produced by the microorganisms. The assay measures the concentration of biocide which is biologically available to react with bacteria rather than providing a chemical determination.

However use of such an assay technique in isolation suffers from the potential difficulty that it only measures the level of biocide available to react with the specific bioluminescent microorganisms used and does not take account of the possible presence of microorganisms in the system which may be resistant to biocide. The biocidal assay is, in accordance with the invention, complemented by an assay for living biomass, which is preferably an assay for the level of ATP (adenosine triphosphate). This gives an overall indication of the level of biomass in the sample and thus confirms whether or not microorganisms are proliferating despite the presence of biocide. These two assays thus provide an enhanced assessment of the water system.

Both assay methods used in accordance with the present invention may be conveniently performed on-site over a short time period. Neither assay requires the use of complex equipment or recourse to complex laboratory procedures. It is moreover possible, and is preferred, to perform the two assays both as assays measuring the level of bioluminescence and to use the same equipment for measuring the level of bioluminescence.

The assay for the concentration of biocide is based upon the reaction of bioluminescent microorganisms, preferably bacteria, with biocide. The level of light produced by the bioluminescent microorganisms after reaction with a water sample containing biocide over a set time period is compared with the light level prior to reaction with biocide. The concentration of biocide in the sample can be determined, by correlation to a standard calibration performed using the make-up water used in the water system containing a known amount of biocide.

Calibration may be performed using a series of samples of known concentration of biocide. The reduction in bioluminescence in the standard samples may be measured for a variety of times and from this, an optimum time can be determined which provides a best linear correlation between reduction in bioluminescence and concentration of biocide. Once such an optimum has been determined, the reduction in bioluminescence need be measured for a sample removed from the water system only over this specific time.

It is important to use a calibration using water sampled from a water system, to take into account the effects of the various components in the water system on the assay.

Such components may for example affect either the biocides in the system or the bioluminescent microorganisms. A calibration performed using water, such as de-ionised water, would not be take these factors into account.

The bioluminescent microorganisms used in the assay may be naturally occurring or may have been genetically modified to provide them with bioluminescence. Examples of types of naturally occurring bioluminescent bacteria which may be used in accordance with the invention include Photobacteria, such as photobacterium phosphoreum or photobacterium fischeri. Alternatively, bacteria such as E. coli may be used which have been engineered so as to express a gene, such as the LUX gene, responsible for bioluminescence. Preferred genetically engineered bioluminescent microorganisms are bioluminescent microorganisms which have been killed, preferably by irradiation. Though such microorganism retain the metabolic function of bioluminescence they do not replicate.Replication can cause complication of the results of the assay and it is also desirable to avoid the use of live genetically engineered microorganisms which may be released into the environment.

Preferably the microorganism is stabilised, for example, by lyophilisation and prior to use is reconstituted using a reconstitution buffer. Such a buffer preferably contains an osmotically-potent non-salt compound such as sucrose, dextran or polyethylene glycol. After reconstitution with buffer the bioluminescent microorganism should be allowed sufficient time prior to use to reach a high level of bioluminescence.

The biocide assay may be used to determine the concentration of a variety of different biocides. Since different biocides will react with the bioluminescent microorganism at different rates, the optimum time between addition of sample and measurement of the reduction of bioluminescence will vary from biocide to biocide and will be determined by a calibration for an individual biocide.

Preferably, the assay will be adjusted so that the reduction in bioluminescence may be optimally measured after a period from 10 seconds to 20 minutes, more preferably 2 to 10 minutes. Where necessary adjustment of reaction times may for example be achieved by dilution of the reaction medium.

Examples of biocides which may be assayed include dibromonitrilo-propionamide, bromonitropropanediol, quaternary ammonium salts, quaternary phosphonium salts, isothiazolinones, glutaraldehyde, methylene bis(thiocyanate) and benzalkonium chloride.

In some cases reaction of biocide with the bioluminescent microorganisms will occur over a period which is too fast to be readily measurable. This is the case for example with halogen biocides such as chlorine or bromine. This effect may nevertheless be used in a system comprising a mixture of fast-acting biocide, such as halogen, and slower-acting biocide by allowing reaction with the fast-acting halogen biocide to occur before the initial level of bioluminescence is measured. In these circumstances reaction with fast-acting biocide is complete before the assay measurement is made and the effect of the fast-acting biocide on bioluminescence is effectively ignored.

The biocide assay may be used to determine the concentration of biocide over a range of concentrations.

Typically the concentration of biocide product will be from 10 to 500, preferably 25 to 250 ppm corresponding to a concentration range of active biocide species from 0.1 to 100, preferably 0.5 to 20 ppm.

The second assay used in accordance with the method of the invention is an assay for the amount of living biomass in the sample. Generally, this will be an assay for the amount of ATP present which gives an indication of the concentration of living biomass. Thus, from this it can be determined whether microorganisms are continuing to persist in the water system despite treatment with biocide.

Assay for ATP is recognised as a measure of living biomass. ATP is the "energy currency" responsible for regulating the activity of many enzymes and it can be readily extracted and assayed with enzyme. The use of ATP assay is well established for variety of microbial enumeration applications. An assay for ATP which is preferably used in accordance with the invention is described in WO 92/12253.

ATP is preferably assayed using the enzyme, firefly luciferase which catalyses conversion of luciferin by ATP to produce light which can be measured in a luminometer.

The amount of light which is emitted is directly proportional to the concentration of ATP extracted from a sample and hence is a measure of the number of cells in the sample. Preferably assay is performed by removing a small, predetermined, amount of sample for example using a sampling stick. This is then treated with an extractant, such as surfactant, to release ATP from the cells in the sample. After a predetermined time, the sample is then transferred to a reaction medium which preferably includes an agent which neutralises the extractant and prevents it interfering with the luciferase reaction. Reagent comprising luciferin and luciferase is then added to the reaction medium, preferably as a lyophilised sample, and after a predetermined time the level of light emitted in the sample is measured.

Such an assay can be performed conveniently using a sampling pen comprising a sampling stick used to retain a small sample. The sample is treated with extractant and is then introduced into a cuvette containing reaction medium.

The reagent containing luciferase/luciferin may be contained in a further compartment and released into the reaction medium. The level of bioluminescence may be measured directly on the reaction medium without removing a sample as the reaction medium is within a cuvette. Since the assay involves introduction of the sample into premade-up reaction medium, the need for any on-site calibration of the assay is avoided. Thus the assay for ATP may be performed conveniently on-site without recourse to complex laboratory procedures.

The level of light produced by the luciferase reaction may be measured using a portable luminometer. The same luminometer may be used, with appropriate control software, to also measure the bioluminescence in the biocide assay.

The method of the present invention may be used to monitor biocide in any type of industrial water system where biocide is used to control the level of microorganisms. It may for example be used to monitor the level of biocide in industrial cooling waters for example in cooling water towers or in closed cooling water systems.

The method has applicability also to water systems used in paper-making and in air-conditioning.

The method can be used to determine biocide in a wide variety of different waters which contain a variety of contaminants and under a variety of conditions. Typically, water used in industrial systems is maintained at a pH from 6.5 to 9.5, more preferably 7.5 to 9.0, i.e. slightly alkaline to prevent corrosion and may contain a variety of additives including corrosion inhibitors, dispersants and polymers. The method of the present invention may be used to assay biocides in the presence of such additives and over such a pH range. The presence of such additives in a particular system and the pH of the water does not adversely affect the assay used in the present invention provided that a standard calibration is performed for the biocide assay using the same water as is used in the system.

Generally, the assays used in the invention will be performed on a sample of water removed from the water system which is allowed to equilibrate with the surrounding atmosphere so as to reach ambient temperature. Preferably therefore the assays will be performed on a sample which is at a temperature from 5 to 30'C.

In accordance with the invention, once the effectiveness of biocide in a system has been determiped, a further dose of biocide may be added to the system in order to maintain the levels of biocide and biomass in the system within predetermined levels. Where biocide is added to the system after monitoring and the initial level of biocide has become depleted typically the same biocide will be added as was initially used, though different biocide may be added. If, however it is observed that there is a proliferation of microorganisms in the water system despite the presence of biocide then a different biocide from that used initially will preferably be used.

The present invention will now be further illustrated by the following Examples: The following figures are referred to in the Examples: Figure 1 shows graphically the results of a calibration for biocide using the LUX biocide reagent assay (Figure la) and the results of assays for biocide and ATP in a cooling tower (Figure lb) described in detail in Example 1.

Figure 2 shows graphically the results of a calibration for biocide using the LUX biocide reagent assay (Figure 2a) and the results of assays for biocide and ATP in a cooling tower (Figure 2b) described in detail in Example 2.

Figure 3 shows graphically the results of a calibration for biocide using the LUX biocide reagent assay (Figure 3a) and the results of assays for biocide using bioluminescence and using a chemical test and assay for ATP in a cooling tower (Figure 3b) described in detail in Example 3.

Figure 4 shows graphically the results of a calibration for biocide using the LUX biocide reagent assay described in detail in Example 4.

Figure 5 shows graphically the results of assays for biocide using bioluminescence and a chemical test and assay for ATP in a cooling tower at tower sampling points (a) and (b) described in detail in Example 4.

Figure 6 shows graphically the results of a calibration for biocide using the LUX biocide reagent assay (Figure 6a) and the results of assay for biocide using bioluminescence and a chemical test and assay for ATP in a cooling tower (Figure 6b) described in detail in Example 5.

EXAMPLES The following assay techniques have been used to measure levels of biocide and living biomass in accordance with the method of the invention: Biocide Assav LUX light reagent (a lyophilised sample of killed genetically modified E.coli in which the LUX gene responsible for bioluminescence is present in a plasmid) was used as a bioluminescent organism. The LUX gene was obtained from Vibrio fischeri as a single continuous Sal 1 fragment wich was inserted into the plasmid vector pACYC 184 and transformed into E. coli, using known techniques described for example by Meighen E.A in Ann. Rev Microbiol., 1988, 42, pplS1-176. The bacteria was killed by irradiation with gamma radiation from a cobalt-60 source (1-8k Gy) but retained bioluminescent properties.Before use the freeze-dried preparation was reconstituted using 0.5 ml of a reconstitution buffer (25mM HEPES, pH 6.0, 75mM NaCl, 12.5mM MgS04).

The organism takes up to 20 minutes after reconstitution to reach its maximum potential bioluminescence. A relatively high light output is required for accurate measurement of high concentrations of biocide. A reading of 10,000 or more on an Amersham portable luminometer is sufficient for the assay.

An initial light reading was taken and then 0.5 ml of sample added. A further light reading after a specific contact time was recorded and the log reduction in light calculated. This provides a measure of the biological efficacy of the biocide. Log light reduction is a reproducible index of biologically available biocide concentration, called the relative biocide unit (RBU). By reference to a calibration, log light reduction can be correlated to a concentration of biologically available biocide. Calibration is performed by a dilution series of known concentrations of biocide using the water from the water system under test. Calibration is performed on-site and compensates for the effect of system water on the bioluminescent reagent and on the activity of the biocide.

Calibration remains valid as long as there are no gross changes in the composition of the water or until a biocide dosing program is altered.

ATP Assay This assay is used to estimate the amount of living biomass in a sample. ATP assays were performed using the Bioscan TM sampling pen available from Grace Dearborn. Using this system a sampling stick is dipped into a test solution and removes a predetermined volume of sample. The sample is contacted with a surfactant composition which extracts ATP from cells in the sample. After a set period the sample is transferred to a cuvette containing a buffered test solution including an agent to neutralise the surfactant.

After a further period freeze-dried reagent, including luciferin and luciferase is added to the cuvette. The light produced after a set period is measured in an Amersham portable luminometer. The level of luminescence, expressed as relative luminosity units (RLU) may be correlated to the concentration of ATP. Specific on-site calibration is not required since the assay uses a cuvette containing a standard buffer solution and does not contain significant amounts of make up water.

EXAMPLE 1 Two cooling towers were examined each of 13.5 m3 capacity.

The sumps of the towers are connected via a weir. The towers were treated with a slug dose of 3 litres of Biomate 5792 (biocidal mixture of chloroisothiazolinone, chloromethylisothiazolinone and glutaraldehyde), to give an estimated concentration of 100 ppm biocide.

A sample of the water was removed from the towers before dosing with biocide and used to prepare a standard calibration for biocide assay using LUX reagent.

Calibration indicated an optimum contact time between addition of sample and measurement of reduction in light level of 3 minutes.

Following dosing, samples were removed for assay of biocide using LUX reagent and of ATP concentration at 20 minute intervals - Standard Calibration for Biocide

Conc Light | Light | Log | Light | Log | Light | Log ppm T=0 T=3min red T=6min red T=9min red 0 40000 14000 0.455 16000 0.397 18000 0.346 31 47000 12000 0.59 10000 0.67 10000 0.67 63 51000 10000 0.71 8500 0.78 6500 0.90 125 55000 7000 0.90 3900 1.15 2400 1.36 250 45000 2900 1.19 1200 1.57 640 1.85 These results are shown graphically in Figure 1(a) , which shows RBU versus ppm (biocide) for a contact tic of 3 minutes.

Tower 1 Time course

BIOCIDE Time after Light Light Conc ATP dosing T=0 T=3min (ppm) (RLU) (hrs) 0:20 38000 3200 202 200 0:40 61000 7600 139 130 1:00 67000 12000 80 190 1:20 57000 10000 84 160 1:40 43000 8100 72 130 2:00 67000 14000 55 77 2:20 51000 10000 66 93 redose 1 litre 2:40 63000 20000 0 85 3:00 57000 7600 128 95 3:20 51000 7800 106 110 3::40 57000 10000 84 98 4:00 49000 7800 98 80 Tower 2

BIOCIDE Time Light Light Conc. ATP (hrs) T=0 T=3min ppm (RLU) 0:20 61000 24000 0 13 0:40 67000 24000 0 57 1:00 67000 22000 0 50 1:20 67000 20000 0 74 1:40 47000 12000 23 90 2:00 1 67000 20000 0 41 2:20 59000 16000 13 53 redose 1 litre 2:40 59000 18000 0 100 3::00 67000 12000 80 60 3:20 59000 12000 60 48 3:40 49000 8500 85 63 4:00 49000 8100 1 96 130 The results for both towers are shown graphically in Figure l(b) which shows ppm biocide/RLU ATP versus time (T). In the figure the solid line ( ) shows the concentration of Stiocide expressed as ppm in tower 1 and the broken line (--) shows the concentration of biocide in tower 2. The levels of ATP, measured as Bioscan RLU, are shown for tower 1(X) and tower 2(1).

The result of biocide assays for tower 1 showed an initial large drop in concentration, prior to a period of slower decline. This drop is predominantly due to the initial biological demand of planktonic organisms. The levels of ATP are initially quite high, but quickly fall, lending support to the idea that the biocide has been "used up" to control planktonic organisms.

1 Litre of biocide was added at 2:20 hours post initial dosing in order to restore the biocide level to 100 ppm of biologically available biocide. A level of 98 ppm was measured following a mixing period of 1:00 hours. This estimate of required biocide was calculated from the results achieved using the LUX biocide assay, and is an example of resetting the biocide level to a pre-determined biologically effective concentration on the basis of the assay results.

Tower 2 showed an increase in ATP levels to a peak at 1:40 hours after dosing. This peak was probably due to the biocide breaking up a film containing microorganisms (biofilm) and releasing more planktonic organisms. The break up of this film could consume biocide from the system and accounts for the low levels of biocide detected initially in tower 2. The level of biocide in tower 2 increased to 96 ppm 1:00 hours after re-dosing, indicating that the initial biological demand had been satisfied.

EXAMPLE 2 Two cooling towers were analysed after dosing with Biomate 743 (containing glutaraldehyde) to give a concentration of approximately 100 ppm of biocide.

A sample of the water was removed from the towers before dosing with biocide and used to prepare a standard calibration for biocide assay using LUX reagent.

The calibration of glutaraldehyde showed a good differentiation of concentrations over the range tested for a contact time of 3 minutes between addition of sample and measurement of reduced light level. After dosing, samples were removed from the tower and assayed for biocide using LUX reagent and for ATP (using Bioscan) at regular intervals. The results of the biocide LUX assay were compared with results of a chemical assay.

Standard Calibration for Biocide

CONCENTRATION 0 3 MINS 5 MINS 9 MINS 31 ppm 55000 12000 11000 8700 Log Reduction 0.661 0.699 1 0.801 63 ppm 55000 9600 7000 3700 Log Reduction 0.758 0.895 1.172 125 ppm 71000 6000 3200 890 Log Reduction 1.073 1.346 1.902 250 ppm 49000 1500 520 100 Log Reduction 1.514 1.974 2.690 500 ppm 40000 200 80 33 Log Reduction 2.301 2.699 3.084 These results are shown graphically in Figure 2(a) which shows RBU versus ppm biocide for contact times of 3 minutes (5), 5 minutes () and 9 minutes (v) and the regression line used (+) Time Course

Tire Light Light Biocide Biocide ATP Bioscan after at TO at T+3 ppm ppm ng/ml RLU dosing (LUX (Chemical (hrs) assay) assay) Pre-dose 49000 14000 0 22.2 0.62 240 0 51000 9400 43 0:30 63000 8000 90 0.29 I 110 0:45 59000 8300 77 177.8 0.10 39 1:00 49000 7800 62 1:15 63000 12000 39 0.29 110 1::30 40000 7400 43 177.8 0.36 140 1:52 36000 8300 < 31 0.23 90 2:30 51000 10000 36 1 0.34 130 These results are illustrated graphically in Figure 2(b) which shows Biocide ppm (0) and Bioscan RLU () versus time (T).

The level of biocide peaked approximately 30 minutes after the end of the dosing cycle. Assuming that the process was active throughout the period, this indicates a mixing time in the system of 45 minutes.

the peak of biocide was associated with a rapid reduction in ATP measured by Bioscan RLU. The biocide peak declined rapidly over the following 2 hours, levelling out at around 30-40 ppm, with a concomitant rise in ATP, indicating that the level and persistence of the biocide does not affect the ATP loading over a prolonged period. In order to reduce the level of ATP further it would be necessary to maintain a higher concentration of biocide over a longer period which could be achieved by increasing the initial dose.

There was a poor correlation between the biocide LUX test and the chemical assay. Throughout the time course, the chemical test producing consistently higher ppm values.

The chemical test showed some level of activity at the predose sample, even though the system had been left without biocide for some time. It should be noted that the chemical test used was not specific for glutaraldehyde and will measure any aldehyde group. It is possible that a contaminant from the cooled process is being measured by the chemical test in addition to biocide.

EXAMPLE 3 A small cooling tower dosed with Biomate 743 (containing glutaraldehyde) was analysed.

Before addition of biocide, a sample of test water was removed and used to prepare a standard calibration for LUX assay of biocide. A contact time between addition of sample and measurement of reduced light level of 6 minutes was selected on the basis of the calibration.

Following dosing, the levels of both biocide and ATP (using Bioscan) were assayed over time at regular intervals. The results of the biocide assay were also compared with a chemical assay for biocide.

Standard Calibration for Biocide

CONCENTRATION O 3 MINS 6 MINS 12 MINS 18 MINS 7.75 ppm 26000 5700 5600 4900 3800 Log Reduction 0.659 0.667 0.725 0.835 15.50 ppm 55000 10000 9800 8500 6400 Log Reduction 0.740 0.749 0.811 0.934 31.00 ppm 57000 8100 6500 3900 2000 Log Reduction 0.847 0.943 1.165 1.455 63.00 ppm 59000 5700 3700 1400 480 Log Reduction . 1.015 1.203 1.625 2.090 125.00 ppm 49000 2300 890 160 48 Log Reduction 1.328 1.741 2.486 3.009 These results are shown graphically in Figure 3(a) which shows RBU versus ppm biocide for contact times of 3 minutes (5), 6 minutes (), 12 minutes (v) and 18 minutes (0) and the regression line used (+).

Time Course

Time Light Light Biocide Biocide ATP Biosca After at TO at ppm ppm I ng/m n Dosing T+6 (LUX (Chemic 1 RLU (hrs) assay) al assay) 0:35 51000 560 > 250(296) 0.26 100 10:50 57000 890 > 250(262) 270 0.10 37 1:05 34000 540 > 250(272) 250 0.12 45 1:20 63000 890 > 250(272) 240 0.10 40 1:35 36000 760 232 1::50 34000 760 228 210 O.11 41 These results are shown graphically in Figure 3(b) which shows ppm biocide measured by LUX assay (0), and chemical assay (+) and Bioscan RLU () versus time (T).

The calibration showed a good differentiation of all concentrations tested. The minimum detection level for gludaraldehyde was less than that obtained using laboratory procedures. This reflects the effect of the tower environment on the activity of the biocide. Detection limits based on laboratory procedures inevitably favour the stability of the organism. Except for cases where inherent toxicity in the system masks the reaction caused by lower levels of biocide, it is expected that the LUX organism will usually find the environment of the water system less favourable than the laboratory conditions used to determine the minimum detection limits. The result of this is that the organism is likely to be more fragile and therefore more susceptible to the effects of the biocide, resulting in an improvement in the minimum detection limit.

This is a good example of the need to calibrate with the product in use, since the material provided for calibration does not contain the same amount of glutaraldehyde as the product dosed. The need to extrapolate the sample results beyond the maximum calibrated concentration is due to this difference, since amendments were made to the calibration based on the expected difference between the calibration material and the dosed product.

Initial high levels of biocide caused a rapid reduction in levels of planktonic organisms as measured using Bioscan RLU. The level of biocide fell slightly, but remained relatively high for 1 hour post dosing. A slight decrease was noted 1 1/4 hours after dosing, and this may reflect the retention time in the system or a demand for cooling placed on the system. The level of ATP reduced and stabilised at a relatively low level indicating that the treatment with biocide was effective.

The LUX results and chemical assay results are in general agreement.

EXAMPLE 4 A cooling tower dosed to approximately 100 ppm Biomate 723 which contains dibromonitrilopropionamide (DBNPA) was analysed. The cooling system was sampled at two points.

The first sample site (HOT) was after the dosing point but before the process water entered the cooling tower. The second sample site (COLD) was after the process water had been cooled in the tower. The HOT sample was warmer than the COLD sample. Both samples were left at room temperature to equilibrate to the calibration temperature before testing.

A sample of water was removed from the tower before biocide dosing and used to prepare a standard calibration for biocide assay using LUX reagent. A contact time of 30 seconds between addition of sample and measurement of reduced light level was selected.

After dosing samples were removed from the tower at regular intervals and assayed for biocide using LUX reagent and for ATP using Bioscan. The results of the biocide assay using LUX reagent were compared using a chemical assay.

Stancard Calibration for fjiocide

CONCENTRATION 0 30 SECS 1 MIN 2 MINS 31 ppm 55000 8400 6100 4300 Dvalue 1 0.6127 1.047 1.807 63 ppm 49000 1400 710 320 Dvalue 0.3238 0.544 0.915 125 ppm 51000 170 160 100 Dvalue 0.2019 0.399 0.739 250 ppm 63000 5.0 Dvalue 0.1219 These results are shown graphically in Figure 4 which sows D-values versus ppm Biocide for contact times of 30 seconds (0), 1 minute () and 2 minutes (v) and the regression line used (+).

Time Course Data for HOT sample site

Time Light Light Biocide Biocide ATP Bioscan After at TO at ppm ppm ng/ml RLU Dosing T+30sec LUX Chemical (hrs) assay Pre 0 1.2 460 0:20 40000 610 84 40 0.67 260 0:30 55000 1100 77 0.57 220 0:40 45000 1200 60 0.81 310 0:50 55000 2300 58 40 1:10 49000 3000 49 0.60 230 1:25 22000 1700 44 0.60 230 1:40 34000 4400 33 0.16 63 1::55 20000 2300 < 31 0.11 44 These results are shown graphically in Figure 5(a) which shows ppm biocide measured iDy LUX assay (O) nd chemical assay (A) and Bioscan RLU (#) versus time (T).

Time Course Data for COLD sample site

Time Light Light Biocide Biocide ATP Bioscan After After at TO at ppm Chemical ng/nl RLU Dosing T+30sec LUX Assay (hrs) assay l 0:20 47000 1500 65 0:30 57000 1700 67 40 1.35520 0:40 45000 1400 66 0.75 290 0:50 43000 1600 61 1:10 28000 1700 50 30 0.26 100 1:25 24000 3800 29 20-30 0.23 89 1:40 39000 3900 33 20 0.31 120 1::55 22000 3400 31 20 0.13 51 These results are shown graphically in Figure 5(b) which shows ppm biocide measured by LUX assay (z) and chemical assay (A) and Bioscan RLU (A) versus time (T).

The hot sample, which is close to the dosing point, showed an initial peak of biocide of 80 ppm. This level fell over the first 20 minutes as the biocide was mixed in the system. The cold point shows an initially lower concentration of biocide than the hot point, due to the usage of biocide in the controlling the tower. After the first 20 minutes both sample sites showed the same levels of biocide, which decreased gradually over the rest of the trial. The ATP levels from both sample sites also showed a similar trend, with levels decreasing throughout the trial.

The biocide treatment is clearly effective, and the levels of biocide remain quire high for long enough to reduce ATP levels.

Chemical estimates for biocide concentration follow the same basic trend as the LUX biocide results, but the absolute values are substantially lower.

EXAMPLE 5 Assays was performed in a cooling tower dosed with 100 ppm of D4469 (10% tetra-alkylphosphonium chloride) and bromine.

Although the biocide assay using LUX reagent can accommodate low levels of oxidising biocide when measuring non-oxidising biocides the halogen was neutralised prior to assay by adding 0.2 ml of 0.1N thiosulphate per 5 ml of water sample.

Prior to adding biocide a sample of water was removed from the system and used to produce a standard calibration for the biocide assay using LUX reagent. A contact time of 12 minutes between addition of sample and measurement of reduced light level was selected as optimal for this system.

Because the biocide has only a sub-lethal effect on the LUX reagent the contact time is selected to ensure that this time is sufficiently short to prevent the recovery of bacteria from the effects of the biocide.

After addition of biocide samples were removed from the system at regular intervals and assayed for biocide using LUX reagent and ATP using bioscan. The results of the biocide assay were also compared to results of a chemical assay for biocide.

STANDARD CALIBRATION FOR BIOCIDE

Concentration j 0 3 mins 12 mins 20 mins 00 ppm 43000 14000 16000 16000 Log Reduction 0.487 0.429 0.429 63 ppm 49000 16000 18000 3300 Log Log Reduction 0.486 0.435 1.172 125 ppm 34000 10000 9000 3300 Log Reduction 0.531 0.577 1.013 250 ppm 79000 20000 9000 6700 Log Reduction 0.597 0.943 1.072 500 ppm 86000 7200 1000 230 Log Reduction 1.077 1.934 2.573 The results of this calibration are shown graphically in Figure 6(a) which shows RBU versus ppm biocide for contact times of 3 mintues (E)), 12 mintues (a) and 20 minutes (v) and the regression line used (#).

TIME COURSE

Time Light Light Biocide Biocide ATP Bioscan after at at pp, ppm ng/m RLU dosing TO T+6 WX Chemical 1 (hrs) Assay Assay Pre- 0.26 100 Dose 1.0 55000 18000 95 0 2.36 910 2.0 43000 14000 95 0 2.10 810 3.0 63000 22000 86 0 2.16 830 4.0 57000 16000 114 1.33 510 These results are shown graphically in Figure 6(b) which shows ppm biocide measured by LUX assay (0) and chemical assay (a) and RLU Bioscan (v) versus time (T).

The toxicity of the bromine to the LUX organism was quite low as shown by the relatively low light reductions produced by the zero concentration in the two calibrations.

The LUX test could have accommodated these levels of bromine present, so the use of thiosulphate to neutralise bromine before the assay at this site was probably unnecessary.

The level of biocide as measured by the LUX reagent in the cooling tower remained close to the expected level of 100 ppm throughout the trial, with an increase at the last time-point.

However, the levels of ATP rose and remained high. The initial rise is likely to be the result of the action of the biocide to disperse film containing bacteria. This sheds doubt on the chemical assay for biocide which failed to detect any biocide in the tower samples. This illustrates the difficulty in relying upon an assay for biocide alone and the advantages of combining this with an assay for ATP.

A further comparison was performed using known dilutions of D4469 made up in de-ionised water and tested with the biocide assay using LUX reagent and the chemical test.

Note the results from the LUX test cannot be compared to the previous calibration due to the differences in the make-up water from the tower and the water used to make the dilutions.

CHENICAL ASSAY

ppm TBDPC (10% Chemical test D4469 active) ppm result (ppm D4469) 125 12.5 T 200 ppm 250 | 25 > 400 ppm 500 50 > 400 ppm LUX ASSAY

Concentration O 3 Mins 12 Mins 125 ppm 51000 10000 6000 Log Reduction 0.708 0.929 250 ppm 65000 9000 2300 Log Reduction 0.859 1.451 500 ppm 77000 I 2900 260 Log Reduction 1.424 2.472 For the assay using the LUX reagent a contact time of 12 minutes on a log light reduction vs. concentration plot was selected, and showed a correlation coefficient of 1.000.

This indicates an excellent differentiation between all concentrations and that the LUX system is working well. The results reported above for the LUX system are thus supported by this calibration. The chemical test produced a result, though the accuracy was poor giving levels significantly higher than expected. The reason for the apparent lack of chemical activity in the process water is unknown though it illustrates the. difficulty in using a chemical test which does not measure the level of biologically available biocide.

Claims (13)

CLAIM8

1. A method of monitoring biocide in an industrial water system, which comprises: (a) assaying the concentration of biocide in a sample removed from the system by contacting the sample with bioluminescent microorganisms and determining from the reduction in the level of bioluminescence, the concentration of biocide; and (b) assaying the level of living biomass in a sample removed from the system.

2. A method according to claim 1 wherein the assay for biocide uses killed bioluminescent bacteria which have been genetically modified to express DNA encoding a gene for bioluminescence.

3. A method according to claim 2 in which the genetically modified bacteria are E. coli which express the LUX gene for bioluminescence.

4. A method according to claim 1 in which the bioluminescence bacteria are naturally occurring.

5. A method according to any one of the preceding claims in which, in the assay for biocide, the level of bioluminescence is measured on contacting the sample and the bioluminescent microorganisms and at a predetermined time from 10 seconds to 20 minutes after contact.

6. A method according to any one of the preceding claims in which the concentration of active biocidal species in the water system is from 0.1 to 100 ppm.

7. A method according to any one of the preceding claims wherein assay for living biomass is an assay of ATP.

8. A method according to claim 7 in which the ATP assay comprises extracting ATP from living cells with extractant, neutralising the extractant, reacting the ATP with luciferin in the presence of a luciferase enzyme to produce bioluminescence and quantifying the amount of ATP from the amount of bioluminescence.

9. A method according to claim 8 in which the levels of bioluminescence in the assays for biocide and ATP are measured using the same luminometer.

10. A method according to any one of the preceding claims in which the sample water assayed is at a pH from 6.5 to 9.5.

11. A method of operating an industrial water system, which comprises: (a) assaying the concentration of biocide in a sample removed from the system by contacting the sample with bioluminescent microorganism and determining from reduction in the level of bioluminescence, the concentration of biocide; (b) assaying the level of living biomass in a sample removed from the system; and (c) maintaining the concentration of biocide and level of living biomass within predetermined limits by adding biocide.

12. A kit for monitoring biocide in an industrial water system which comprises: (a) means for assaying the concentration of biocide in a sample removed from the system by contacting the sample with bioluminescent microorganism and determining from the reduction in the level of bioluminescence, the concentration of biocide; and (b) means for assaying the concentration of living biomass in a sample removed from the system.

13. A kit according to claim 12 which comprises: means for assay of biocide comprising lyophilised bioluminescent microorganisms and reconstitution buffer; means for ATP assay comprising luciferin and luciferase; and means for measurement of bioluminescence in both assays and optionally control software to correlate the level of luminescence with the concentration of biocide and ATP.