BACKGROUND OF THE INVENTION

This invention relates generally to chemical analysis and more particularly to a quick, non-boiling method to determine polyphosphate or phosphonate concentration in an aqueous sample.

DESCRIPTION OF RELATED ART The determination and control of chemicals such as salts, minerals, metals, etc. in water treatment systems is important to industrial, commercial, municipal, home and other water users. The use of inorganic phosphate compounds and phosphonates is particularly important and common. Phosphorus compounds have been utilized in the water treatment industry for many years. Orthophosphate (P0 ₄ . ₃ ) is the simplest form of phosphate and is commercially available as phosphoric acid, and mono-, di-, and trisodium phosphate. It is an excellent corrosion inhibitor and is used, for example, in boiler water treatment systems. Polyphosphates are polymeric forms of inorganic phosphate. Orthophosphates and polyphosphates are inorganic phosphates. The more commonly available polyphosphates are tetra-potassium pyrophosphate (K ₄ P ₂ 0 ₇ ) , sodium tripolyphosphate (Na ₅ P ₃ Oj ₀ ) , sodium hexametaphosphate, and other salts thereof. Generally, pyrophosphate, tripolyphosphate, andhexametaphosphate are the

most common polyphosphates used in water treatment systems. Other polyphosphates include glassy phosphate (NaP0 ₃ ) ₂ ι, (NaP0 ₃ ) , (NaP0 ₃ ) ₉ , (NaP0 ₃ ) ₅ , trimetaphosphate (NaP0 ₃ ) ₃ , sodium pyrophosphate (Na ₂ H ₂ P ₂ 0 ₇ ) , and another tripolyphosphate (Na ₃ H ₂ P ₃ O, ₀ ) . These polyphosphates have many uses in water treatment systems as discussed below. The primary use of polyphosphate in industrial water treatment is for scale inhibition, corrosion inhibition and iron/manganese stabilization at 3 - 6 pp as P0 ₄ . At higher levels polyphosphates are good detergents and suspension agents. Another major water treatment application for polyphosphates is in open recirculating and in once-through water systems for corrosion inhibition or scale control in industrial and potable water systems. Polyphosphates are also used in open recirculating and once-through cooling water systems. The most common and largest dollar value industrial once-through cooling and process applications are in the paper and steel industries where corrosion control and iron stabilization are needed. In boiler water applications, polyphosphates hydrolyze to form orthophosphate. In cooling water systems, some hydrolysis (reversion) of polyphosphate to orthophosphate will take place, depending on the presence of bacteria, pH, bulk water temperature, and the skin temperature of the heat transfer surfaces. Phosphonates are organic phosphorus compounds which have a carbon to phosphorus bond which gives them greater stability

against oxidation. Commonly used phosphonates in the water treatment industry include AMP (amino methylene phosphonic acid or nitrilo tris (methylene phosphonic acid) ; HEDP (hydroxyethylidene diphosphonic acid) ; PBTC (phosphonobutane tricarboxylic acid) ; HPA (hydroxyphosphonoacetic acid) ; and HDTMP (hexamethylene diamine[tetra] methylene phosphonic acid) . These compounds are utilized primarily as scale control agents and calcium carbonate scale inhibitors in cooling and boiler water applications. They are particularly useful in cooling water treatment because of their resistance to degradation at cooling water temperatures. Also, phosphonates tend to be resistant to microbiological oxidation — a constant concern in open recirculating systems. Since their introduction to water treatment some 20 years ago, the major difficulty in their application has been product control and water analysis. Although these phosphonates are relatively stable to hydrolysis, they can be converted into orthophosphate in the presence of oxidizing agents, such as ozone, chlorine or bromine. It is generally preferable to maintain polyphosphates and phosphonates in their form as polyphosphates and phosphonates in water treatment systems rather than have them break down or revert into orthophosphate. Orthophosphate is effective in certain respects but tends to be more likely to form calcium phosphate scale.

Depending on the hardness of the water and other factors, it is generally desirable to maintain a certain or preselected or predetermined concentration of orthophosphate in the water, as well as a certain or preselected or predetermined concentration of polyphosphates and/or phosphonates in the water. Generally, the harder the water, the less orthophosphate you want in the water. Polyphosphates tend to revert to orthophosphate. Phosphorus compounds can enter a water system through such things as makeup water, process contamination, and airborne sources. Thus, in order to maintain the proper concentrations of phosphorus compounds in the water system, it is important to be able to test for orthophosphate concentrations as well as polyphosphate and phosphonate concentrations, so one will know how much of which compound to add. A treatment program may, for example, comprise adding or maintaining preselected levels of orthophosphate and/or pyrophosphate and/or tripolyphosphate and/or hexametaphosphate and/or phosphonates. With regard to polyphosphates, particularly pyrophosphate, overfeed is expensive and can cause scaling and underfeed can cause production problems and equipment degradation or eventual loss due to increased corrosion rates. With regard to phosphonates, overfeed can cause hard, tightly adherent calcium phosphonate to form; underfeed can result in calcium carbonate scale formation. Both these conditions can impact production, heat exchange efficiency and ultimately, in industrial situations, plant profitability. In some

chemical treatment formulations, extra phosphonate is added solely to allow for easier testing of the phosphonate concentration, an expensive and unadvisable technique. Accordingly, it is important to constantly or regularly monitor the concentration of orthophosphate, polyphosphates, and phosphonates in water treatment systems to maintain their concentration in the water within predetermined levels. This requires water analysis techniques to measure or determine the concentrations. With regard to polyphosphates, the prior art test procedures require the use of boiling with acid to hydrolyze polyphosphate to orthophosphate. These prior art test procedures, such as those described in Standard Methods for the Examination of Water and Waste Water, prepared and published jointly by American Public Health Assn. , American Water Works Assn. , and Water Pollution Control Federation, 17th Edition, Editors: Lenore S. Clesceri, et al., Washington, D.C. (1989) Part 4000 pp 166-181, and in ASTM Method No. D515-88 (1988) , and such as those utilized by Nalco Chemical Company, Grace Dearborn (W.R. Grace & Co.) , The Mogul Corporation dba Dexter Water Management Systems Division of The Dexter Corporation, and Hach Company (the primary supplier of test kits to the water treatment industry) , are well known in the art and are incorporated herein by reference. Basically, the procedure is as follows. First, the concentration of orthophosphate is measured. It involves a two step process: a) reaction of the orthophosphate with molybdate in the presence of a catalyst to form a

phosphomolybdate complex (H ₂ PMθ _j2 O ₄₀ )"; and b) the phosphomolybdate complex is then reduced by a reducing agent such as ascorbic acid causing the formation of a characteristic molybdenum blue species. The intensity of the blue color is then determined using a color disc comparator or, for greater accuracy, a colorimeter or spectrophotometer. The intensity of the blue color is correlated with the concentration of orthophosphate. Other colorimetric and non- colorimetric quantitation methods as are known in the art can also be used. Next, the polyphosphate is converted into orthophosphate, the concentration of all orthophosphate in the sample (both that present originally and that produced by conversion of polyphosphate into orthophosphate) is measured using the procedure described above, the initial reading or measurement for orthophosphate is subtracted from the second to obtain the difference, then that difference in orthophosphate readings is correlated with the concentration of polyphosphate present. The polyphosphate is converted into orthophosphate by boiling with, for example, sulfuric acid for 30 minutes. There are several prior art test procedures which are known in the art for determining the concentration of phosphonates in water samples. These procedures are incorporated herein by reference and include oxidizing acid digestion (see Standard Methods (cited above) , Part 4000, pp 166-181) , ultraviolet photochemical oxidation method, and microwave oxidation digestion. As with the polyphosphate

methods, these methods rely upon an initial measurement of orthophosphate present, a conversion of phosphonate to orthophosphate, a second measurement of orthophosphate, and subtracting the first reading from the second reading to show the concentration of phosphonate present. Orthophosphate is generally measured using a colorimetric method as described earlier. Phosphonates are organic compounds having the form R- P0 ₃ H ₂ . More specifically they have a carbon to phosphorus bond (C-P0 ₃ H ₂ ) . It is necessary to oxidize the phosphonates in order to break this carbon to phosphorus bond and generate orthophosphate. In the presence of excess oxygen (by the addition of potassium persulfate) , this oxidation can proceed by the action of boiling, UV light, or microwave radiation. When the oxidation is carried out in the absence of acid, no significant hydrolysis of the polyphosphates occurs, making this procedure a true test for phosphonates. In the presence of acid and an oxidizer, the polyphosphates and phosphonates will be converted to orthophosphate and the procedure will detect the total phosphorus content as phosphate (P0 ₄ ) . In the microwave procedure, the sample is placed in a microwave oven, and microwave energy is used to break the carbon-phosphorus bond and produce orthophosphate. The microwave procedure will not work on inorganic polyphosphates in the absence of acid. With regard to polyphosphate and phosphonate water treatment programs, daily or other regular testing is

desirable to maintain the phosphate concentration within predetermined levels. However, the methods of the prior art are difficult, sometimes dangerous, and time consuming, and consequently, are often not done. Polyphosphate determination requires time-consuming boiling with acid. With regard to phosphonates, chromate or molybdate is sometimes added to permit easier analysis and monitoring, but these present cost and environmental concerns; the thorium nitrate test is partially successful but is subject to severe interferences such as ortho P0 ₄ - and thorium is radioactive and is an environmental and handling concern. The thorium nitrate test forms a complex without breaking the carbon-phosphorus bond. The microwave and ultraviolet methods require expensive equipment, require an electrical power supply, are not readily portable, and may present environmental and handling concerns.

Accordingly, there is need for a cheaper, simpler, and easier method for polyphosphate and phosphonate determination. That is provided by the present invention, which utilizes an enzymatic method for polyphosphate and phosphonate determination. The enzymatic method does not use acid, does not require boiling, requires equipment which is much less expensive and much easier to handle than the boiling digestion, microwave, or ultraviolet procedures. There is much less, if any, environmental concern using the enzymatic method. Also, the enzymatic procedure is much faster compared to the boiling acid digestion method. The advantages of the

enzymatic procedure are used to provide a self-contained test kit which is portable and enables repetitive field tests, for example, up to fifty on-site tests at plants or facilities having water treatment systems. It is believed that enzymes such as those disclosed herein have been used for many years in connection with enzyme kinetic studies.

SUMMARY OF THE INVENTION There is disclosed a method for determining or measuring the amount or concentration of orthophosphate precursor present in an aqueous sample. As used in the specification and claims, orthophosphate precursor is a compound from which orthophosphate can be obtained by hydrolysis or oxidation, such as by hydrolyzing pyrophosphate or tripolyphosphate or other polyphosphates, or oxidizing phosphonates, to orthophosphate, or mixtures of such compounds. The method includes treating the aqueous sample by contacting it with an effective amount of phosphatase to cause said orthophosphate precursor to yield orthophosphate in the treated aqueous sample, measuring the concentration of orthophosphate present in the treated aqueous sample, and utilizing the measurement obtained in the measuring step in determining the concentration of orthophosphate precursor present in the aqueous sample. Typically the aqueous sample will contain orthophosphate, and the concentration of orthophosphate in the

aqueous sample is determined and accounted for in determining the concentration of orthophosphate precursor present in the aqueous sample. A portable test kit for performing phosphate analysis in aqueous samples is also disclosed. The test kit includes a container of phosphatase and means for measuring the concentration of orthophosphate present in an aqueous sample. The means can include a color comparator. Other means include a portable spectrophotometer, and a filter photometer.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a perspective view of a portable test kit according to the present invention.

FIG. 2 shows a perspective view of means for measuring the concentration of orthophosphate in an aqueous sample.

DESCRIPTION OF THE PREFERRED EMBODIMENT The inventors have found that enzymes can be used to convert polyphosphates and phosphonates to orthophosphate in connection with the determination of polyphosphate and phosphonate concentrations in water samples. The procedure for determining polyphosphate and phosphonate concentrations in water samples is basically the same as that utilized in the prior art (see, for example, the references cited above) ,

except that enzymatic means are substituted for boiling with acid, UV light, and microwaves, for converting polyphosphates and phosphonates to orthophosphate in the test protocol. Preferred embodiments of the invention are disclosed in the following Examples. If and when required, the samples were appropriately diluted to bring them within the range of the instruments used. In the practice of the present invention, it may be necessary or desirable to dilute the sample being measured to bring it within the range of the instrument or instruments being used. The enzymatic method of the present invention is particularly effective for determining polyphosphate, particularly pyrophosphate, concentrations in aqueous samples in the range of zero to fifty mg/1 as P0 ₄ , subject to the limitations of the colorimetric quantitation method utilized.

EXAMPLE 1 An aqueous sample from a customer was pbtained. The only phosphorus compounds the customer used were pyrophosphates (principally tetrapotassiumpyrophosphate) thus thephosphorus compounds the sample was believed to contain were only or principally orthophosphate and pyrophosphate, although other polyphosphates may have been present. First, an analysis utilizing the methods of the prior art was performed to determine the concentration of orthophosphate. Briefly, one PhosVer 3 Phosphate Reagent powder pillow from Hach Company,

P.O. Box 389, Loveland, CO 80539, sized for a 5 ml. sample, was mixed with a 5 ml. sample in a test tube and permitted to react for 2 minutes, during which a blue color appeared. (The Hach PhosVer 3 Phosphate Reagent powder pillow contains less than 85% potassium pyrosulfate [7790-62-7]; less than 25% L- ascorbic acid [50-81-7]; less than 5% sodium molybdate [7631- 95-0]; and less than 1% each of other components.) This blue sample tube, along with a blank (tube with sample water, but without the powder pillow added) were inserted in a color comparator, known in the art and available from Hach Company. The disk was rotated to obtain a color match. The reading on the scale indicated a concentration of orthophosphate of 17 mg/1 (as P0 ₄ ) . Second, 5 ml. of sample water, diluted as required, was placed in a container. To this was added 5 units (0.1 ml. of diluted suspension) of Product No. P-1435 from Sigma Chemical Co. P.O. Box 14508, St. Louis, MO 63178. Product P-1435 is an acid phosphatase (Orthophosphoric-monoester phosphohydrolase [acid optimum]; EC 3.1.3.2); Type XA: From Sweet Potato; CAS [9001-77-8]. One unit of this product will hydrolyze 1.0 micromole of p-nitrophenyl phosphate per min. at pH 4.8 at 37°C. At a level of 5 units, no denaturing or filtering of the enzyme was required since the concentration of the enzyme was insufficient to interfere in the subsequent test. The sample, with sweet potato phosphatase added, was swirled to mix. At this time the enzyme hydrolyzed the polyphosphate to orthophosphate. No waiting time was required

or utilized. Without waiting, the contents of a Hach PhosVer 3 Phosphate Reagent powder pillow for a 5 ml. sample was added as described above, and the procedure described above was thereafter followed. (Enzyme was not added to the blank, since addition of the enzyme to the sample at this concentration did not impart any color) . The reading indicated a concentration of orthophosphate of 140 mg/1 (as P0 ₄ ) . This would be the orthophosphate present originally plus the orthophosphate produced when the enzyme hydrolyzed the polyphosphate to orthophosphate. By subtracting 17 from 140, it was determined that there were 123 mg/1 (as P0 ₄ ) of polyphosphate present in the sample. The procedure of Example 1 described above was carried out at room temperature and without boiling; no heating was required or utilized and no filtration after addition of the enzyme was necessary or utilized. The sample of Example 1 was also tested using the prior art technique of boiling with acid (basically as outlined in Standard Methods _f cited above) to convert or hydrolyze the polyphosphate to orthophosphate. Sulfuric acid was used with persulfate and the sample was boiled for 30 minutes under pressure from 98 to 137 kPa. A colorimetric method utilizing a Hach PhosVer 3 powder pillow and a DR-2000 spectrophotometer was used to determine phosphate concentration. This method determined orthophosphate concentration of 15 mg/1 (as P0 ₄ ) ; orthophosphate plus polyphosphate concentration of 137 mg/1

(as P0 ₄ ) ; and, by subtraction, polyphosphate concentration of 122 mg/1 (as P0 ₄ ) . Comparing the results using enzyme hydrolysis with prior art acid boiling hydrolysis, it can be seen that the enzyme method of the present invention gave almost identical results.

EXAMPLE 2 The procedure utilized in Example 1 was used on a second sample from a customer where the only phosphorus compound the customer used was pyrophosphate. The results were as follows and are expressed in terms of mg/1 as P0 ₄ . Boiling with Acid Method Enzyme Hydrolysis Method orthophosphate 11 13 orthophosphate plus polyphosphate 16 17 polyphosphate 5 4

The results are sufficiently close so as to be commercially useful. The differences can be attributed, at least in part if not wholly, to the fact that the colorimetry of the boiling method was done with a DR-2000 spectrophotometer rather than a hand-held comparator, and the difference between 4 and 5 on a hand-held comparator is very slight. This is well within the range of accuracy required for purposes of monitoring commercial water treatment systems.

EXAMPLE 3 Tests were run utilizing sweet potato phosphatase (Product P-1435) and another phosphatase, an inorganic pyrophosphatase (pyrophosphate phosphohydrolase) from baker's yeast, specifically Sigma Product No. 1-4503; (EC 3.6.1.1); CAS [9024-82-2]. A sample containing tetrapotassium pyrophosphate was prepared. No breakdown of pyrophosphate to orthophosphate was detected, prior to hydrolysis. Analysis utilizing the boiling with acid technique for 40 minutes without pressure showed a concentration of pyrophosphate of 4.0 mg/1 (as P0 ₄ ) . Analysis utilizing the method and enzyme of Example 1 was conducted, showing a concentration of pyrophosphate of 4.6 mg/1 (as P0 ₄ ) . Analysis utilizing the method of Example 1 was also conducted, except that the enzyme used was Product 1-4503 and 10 units were used. The result showed a concentration of pyrophosphate of 4.9 mg/1 (as P0 ₄ ) . These results are close enough for commercial purposes, as described in Example 2.

EXAMPLE 4 A test was run to determine if the enzymatic hydrolysis method would work on sodium tripolyphosphate. A sample of sodium tripolyphosphate was prepared. Analysis by means of the boiling with acid technique (as described above) was conducted, and showed a concentration of tripolyphosphate of

3.4 mg/1 (as P0 ₄ ) . The sample was also checked utilizing the procedure and enzyme utilized in Example 1, except that the 5 units of sweet potato phosphatase was permitted to react with the sample for one minute. The result showed a concentration of tripolyphosphate of 3.0 mg/1 (as P0 ₄ ) . Additional testing showed that approximately 1 minute was required to obtain adequate recovery, and that additional time delay did not increase the yield. The difference between 3.0 and 3.4 mg/1 is not deemed significant, particularly since the margin of error on the hand-held comparator is at least 10%.

EXAMPLE 5 Tests were run to determine if the enzymatic method would work on sodium hexametaphosphate. A sample of sodium hexametaphosphate was prepared. Analysis by means of the boiling with acid technique showed a concentration of hexametaphosphate of 3.2 mg/1 (as P0 ₄ ) . In both samples in this Example, the orthophosphate in the original sample was below detection limits. The sample was also checked utilizing the procedure and enzyme utilized in Example 1, except that the 5 units of sweet potato phosphatase was permitted to react with the sample for different time periods. A two minute time delay or waiting period resulted in a reading for the concentration of hexametaphosphate of 1.5 mg/1 (as P0 ₄ ) . A five minute time delay resulted in a reading of 1.8 mg/1 (as P0 ₄ ) . Longer time delays out to 20 minutes did not increase

the yield. This testing procedure will work adequately with respect to hexametaphosphate if the time delay is 2 - 5 minutes and the result is multiplied by 2. This was confirmed by a subsequent test on a sodium hexametaphosphate sample determined by the boiling with acid technique to contain 6.0 mg/1 (as P0 ₄ ) of hexametaphosphate. The test was as above, utilizing the same sweet potato phosphatase with a time delay of two minutes. The result was a reading of 3.0 mg/1 (as P0 ₄ ) of hexametaphosphate, 1/2 of the 6.0 reading. A short waiting period, such as five minutes or less, preferably two minutes or less, is preferable, since the speed with which the test can be conducted makes it more useful.

EXAMPLE 6 Tests were run to determine enzymatic activity towards the oxidation of phosphonates to orthophosphate. A sample of amino-(tris)-methylene phosphonate (AMP) was tested. It showed 2.0 mg/1 (as P0 ₄ ) upon analysis with the microwave technique, which is known in the art. The sweet potato phosphatase (P-1435) showed no activity towards AMP; however, an indeterminate amount (more than 5 units) of Sigma Chemical Co. Product P-3627 acid phosphatase, Type I: From Wheat germ (contains lipase) was added to AMP. The large amount of enzyme turned the sample yellow. The sample was then allowed to sit at ambient temperature for 15 minutes. The contents of a standard Hach powder pillow was then added

and a blue color developed. However, the underlying yellow color combined with the blue to turn the sample green, precluding quantitative colorimetric analysis with the blue comparator. However, a spectrophotometer could be set to a wavelength which would filter out the yellow component and measure only the blue component, thus permitting quantitative analysis. Moreover, it is believed that well known testing and graphing techniques could be used to develop a correlation chart, charting color against known concentrations. Such a correlation chart would permit the use of Product P-3627, via the enzymatic procedures described above, to determine the concentration of AMP in a sample via a hand-held comparator. An indeterminate amount (more than 5 units) of Product P-3627 was added to an approximately 6 ppm sample of hexamethylene diamine tetra (methylene) phosphonate (HDTMP) and let stand at ambient temperature for 16 hours. The amount of enzyme turned the sample yellow, so that when the powder pillow was added, the blue color combined with the yellow to turn the sample green, precluding colorimetric quantitation with the hand-held blue comparator. For the reasons set forth above, it is believed that Product P-3627 could be used to determine the concentration of HDTMP in a sample. It is believed that other enzymes may be found which have adequate activity towards the various phosphonates to permit their colorimetric quantitation, preferably with the hand-held comparator known in the art.

Five units of Product P-3627 (approximately 8.3 mg of powdered enzyme) was added to a 5 ml. sample of tetrapotassium pyrophosphate. A five minute waiting period at ambient temperature was utilized. The sample turned yellow, and then green when the powder pillow was added. The comments in the preceding paragraph again apply.

EXAMPLE 7 A test was run utilizing an alkaline or base phosphatase, specifically Sigma Product No. P-5521; (EC 3.1.3.1) ; Type VII- S: From Bovine Intestinal Mucosa; CAS [9001-78-9] . A sample containing tetrapotassium pyrophosphate was prepared. Analysis utilizing the boiling with acid technique showed a concentration of pyrophosphate of 3.1 mg/1 (as P0 ₄ ) . Analysis utilizing the method of Example 1 was also conducted, except that the enzyme used was Product P-5521, an indeterminate amount of that enzyme was used, and it was permitted to react for 5 minutes. The result showed a concentration of pyrophosphate of 3.4 mg/1 (as P0 ₄ ) . A sample of sweet potato phosphatase (Product P-1435) was allowed to sit at room temperature for about 5 months. It was then tested and found to retain its activity. Thus the enzyme is believed to be sufficiently stable for test kit purposes. Routine test procedures may be used to identify other phosphatases which would work effectively in the practice of the present invention. For example, known phosphatases can

be tested to identify those effective at hydrolyzing, oxidizing, or otherwise breaking down preselected polyphosphates or phosphonates to yield orthophosphate. Also, well-known testing procedures can be utilized to identify and isolate additional phosphatases which are effective at breaking down particular or preselected polyphosphates and phosphonates to yield orthophosphate. For example, a nutrient medium can be prepared in which the only orthophosphate source is a particular polyphosphate or phosphonate, for example, PBTC. Then a wide spectrum of bacteria, etc. can be placed in the medium to select out those which can survive and grow in the medium. A bacterium which is thus selected can then be grown in quantity and processed to extract and isolate the phosphatase responsible for breaking down the polyphosphate or phosphonate in question (here, PBTC) to yield orthophosphate. No buffers (other than what may have been present in the enzyme suspension) were used or added in any Examples; it was not necessary to adjust the pH in any Examples. In some cases it may be necessary to use or add buffers or to adjust the pH. Also, it may be useful to select an enzyme (for example, an alkaline phosphatase) based on the pH of the sample (in this case, alkaline) . Generally greater concentrations of enzyme can be used for shorter periods, and lesser concentrations can be used for longer periods, so long as greater concentrations do not constitute an interference necessitating filtration or impart color which would interfere with colorimetric

quantitation or make it more difficult. Lesser concentrations are preferred, principally since less color is imparted. It is believed that any colorimetric quantitation method as known in the art can be used with the disclosed enzyme procedure. Several of these colorimetric methods are described in Standard Methods, cited above, and are incorporated herein by reference. These methods include the vanadomolybdic acid method, which is most useful for routine analyses in the range of 3 to 60 mg/1 as P0 ₄ , the stannous chloride method, which is more suited for the range of 0.03 to 15 mg/1 as P0 ₄ and the ascorbic acid method, which is more suited for the range of 0.03 to 50, and more preferably, 0.03 to 15, mg/1 as P0 ₄ . At concentrations in excess of 50, eg, 60 - 70, mg/1 as P0 ₄ , the ascorbic acid colorimetric method becomes subject to inaccuracies due to deviations from Beer's Law. Other non- colorimetric methods can also be used to determine orthophosphate concentration, including the inductively- coupled plasma method, nuclear magnetic resonance, and gravimetric methods. Generally, in order for an enzyme to be active or effective with regard to its substrate, the presence of one or more cofactors is required. For example, sweet potato phosphatase generally requires the presence of magnesium as a cofactor in order to be effective. Other common cofactors include calcium, iron, and molybdenum. Cofactors as are known in the art may be useful or required in the practice of the present invention. The enzyme preparations described herein

came premixed with the appropriate cofactors necessary for proper activity. FIG. 1 shows a preferred portable test kit 10 according to the present invention, the test kit being easily portable in one hand. The test kit has a carrying case 12 within which is contained an eye dropper 14, two test tubes 16 with stoppers 18, a bottle 22 containing enzyme such as phosphatase, which can, for example, be powdered or a liquid suspension, a bottle 24 containing a reagent composition such as Hach PhosVer 3 powder pillows or other reagents for colorimetric quantitation, and a color comparator 20. The eye dropper can be used to help fill the test tubes with samples, water, and/or enzyme. The sample is measured for orthophosphate concentration to establish a baseline, as described above. The enzyme and powder pillows are added to the contents of the test tubes and mixed sequentially, and the samples are read in the color comparator, as shown in FIG. 2. FIG. 2 shows a color wheel 28 in a case 29 of the color comparator 20. The blank test tube 16A and the sample test tube 16B are placed in the comparator and viewed in strong light through the openings 30, 32. The color wheel is turned until a color match is achieved, at which point a reading on the color wheel is taken through the opening 26, which correlates the strength of the blue color to the concentration of orthophosphate in the sample. Once the phosphate level in the water treatment system is determined utilizing the present invention, steps which are

known in the art can be taken to adjust the level or bring the level within a predetermined range and/or to maintain or control the level within a predetermined range. Frequently polyphosphate will be fed into the system at a predetermined treatment or feed rate to achieve the predetermined treatment concentration. The enzymatic procedure of the present invention can also be utilized (a) in quality control laboratories to determine polyphosphate and phosphonate concentrations in production samples from such products as phosphate concentrates, phosphate additives, etc., and (b) to test for phosphate and phosphonate concentrations in water samples where phosphorus compounds are added to water systems to test for such process variables as volume of vessel or system, bleed rate, and to act as a tracer so the concentration of another additive, such as a polymer, which is hard to test directly, can be monitored. To calculate the volume of a vessel or system filled with water, the concentrations of phosphorus compounds are initially measured to establish a baseline. Then a known quantity of a phosphorus compound, such as pyrophosphate, is added and permitted to mix. Then the concentrations are remeasured. Using this information, the volume of the vessel or system can be calculated. For example, if the baseline shows no phosphorus compounds, and 1.4 kg. of tetrasodium pyrophosphate (71.4% P0 ₄ ) is added to the system, and after it is allowed to mix, a sample is drawn from the system and

found, via the enzymatic procedure, to contain 1 mg/1 P0 ₄ , the volume of the system is 1 million liters. The bleed rate of a system (the rate at which water leaks out of the system) can be calculated using a similar methodology. If a polymer, such as polyacrylate, and a phosphorus compound, such as pyrophosphate, are added to a water system in a known ratio and there is no chemical change in the additives, the concentration of the polymer can then be determined by measuring the concentration of the pyrophosphate. In this way the pyrophosphate acts as a tracer to a compound to be traced. Phosphorus compounds are particularly useful for all the above procedures since they are nontoxic when added properly, relatively inexpensive, easy to use, and frequently must be added anyway for corrosion and scale inhibition. Previously, chromium, lithium, molybdate, organic dyes, etc. have been tried for volume of vessel, bleed rate, and tracer applications, but these approaches are less useful than those described above because they have problems such as toxicity, degradation, expense, and testing difficulties. Although the preferred embodiments of this invention have been shown and described, it should be understood that various modifications may be resorted to without departing from the scope of the invention as disclosed and claimed herein.