Enzymatic analysis of urea in swimming pool water

Enzymatic analysis of urea in swimming pool water — summary

Significance of the topic

Urea and other nitrogenous contaminants introduced by bathers (urea, ammonia, amino acids, creatinine, uric acid) react with free chlorine to form chlorinated by-products and chloramines that impair hygiene and disinfection in swimming pools. Accurate monitoring of urea is therefore important to manage chloramine formation risk and microbial nutrient load. Finnish regulation (Valvira) sets an upper limit of 0.8 mg/L urea in pool water, creating a need for sensitive and specific analytical methods compatible with routine laboratory workflows.

Objectives and study overview

This application note evaluated an enzymatic urea determination method (urease + glutamate dehydrogenase, GLDH) implemented on Thermo Scientific discrete analyzers (Gallery and Aquakem). Goals were to demonstrate method principles, automation capability, analytical performance (limit of detection, precision, systematic error), and method correlation vs. the chemical Koroleff persulfate digestion method and an independent enzymatic reference laboratory.

Methodology and reagents

Principle

• Urease hydrolyzes urea to ammonium and bicarbonate.
• GLDH consumes 2-oxoglutarate and NH4+ with concomitant NADH oxidation to form L-glutamate; NADH consumption at 340 nm is measured.
• Urea concentration is reported as the difference between total ammonia after urease treatment (u rea-derived ammonia plus free ammonia) and free ammonia measured separately. Thus the test is effectively calculated from two measurements.

Sample handling and pre-treatment

• Residual free chlorine interferes with enzymatic assays; sodium thiosulfate dechlorination is recommended and was applied automatically in the discrete analyzers.
• Samples were analyzed within 48 hours of collection.

Assay sequence (Aquakem discrete analyzer example)

• Urea assay: dispense 80 µL sample, add 3 µL dechlorination reagent (R4), add 40 µL R1 and 10 µL R2, incubate 300 s, take blank, add 10 µL R3, incubate 900 s, measure absorbance at 340 nm. Calibration uses a 6 mg/L stock automatically diluted; polynomial calibration fit reported.
• Ammonia assay (low-range ~500 µg/L): dispense 100 µL sample, add 15 µL R1, blank, add 15 µL R2, incubate 540 s, measure at 660 nm. Reaction follows salicylate/sodium nitroprusside chemistry at pH ~8; calibration from a 2 mg/L stock solution (automated dilutions).

Used instrumentation

• Thermo Scientific Gallery discrete analyzer (automation of reagent additions, incubation, blanking, photometric detection).
• Thermo Scientific Aquakem discrete analyzer (described testflows and automation).
• Photometric detection at 340 nm for NADH-based GLDH readout; 660 nm for salicylate-based ammonia assay.

Main results and discussion

Analytical performance

• Determination limit: measured with 0.1 mg/L control (n=25) producing results 0.080–0.130 mg/L; detection limit set at 0.064 mg/L.
• Systematic error: for 1.0 mg/L controls, error across multiple reagent lots and calibrations ranged 0–18% with mean ~6.5%. For low-level controls (0.2 mg/L) average error was higher (~18.1%), attributed to compounded uncertainty from the calculated test (subtracting free ammonia) and high sensitivity of ammonia chemistry to atmospheric contamination.
• Throughput and automation: method readily automated; typical reporting capacity exceeds 100 results per hour on discrete analyzers.

Method comparison and correlation

• Two enzymatic implementations compared (Thermo Scientific method vs an enzymatic reference laboratory) showed excellent correlation (r2 ≈ 0.995).
• The Koroleff persulfate digestion (chemical) method correlated less well with the enzymatic methods (r2 ≈ 0.789) and tended to give lower recoveries for higher urea concentrations. Example: sample with ~1.9–2.1 mg/L by enzymatic methods returned ~0.5–0.92 mg/L by Koroleff.
• Enzymatic method performance in an external proficiency test produced results close to expected values for synthetic and real pool samples (all proficiency results rated Excellent in the reported test set).

Sources of error and limitations

• The enzymatic workflow yields a calculated urea value (total ammonia after urease minus free ammonia); using two independent chemistries increases propagated error, particularly at low concentrations.
• Ammonia measurements are sensitive to atmospheric contamination; strict handling and blanking are essential for low-level accuracy.

Benefits and practical applications

• Higher specificity for urea compared with classical chemical digestion (Koroleff) and generally improved accuracy, especially at elevated urea levels.
• Low achievable detection limit (~0.064 mg/L) suitable for routine regulatory monitoring (Valvira limit 0.8 mg/L in Finland) and early detection of contamination trends.
• Full automation on discrete analyzers reduces manual handling, increases throughput (>100 results/h) and improves reproducibility for laboratory and routine QA/QC environments.
• Prepackaged ready-to-use reagents simplify implementation across different laboratories.

Future trends and potential applications

• Direct single-chemistry urea assays (avoiding calculated subtraction) or integrated reagent formulations could reduce low-level uncertainty.
• Online or near-line monitoring systems for pools and public facilities integrating automated enzymatic assays would enable real-time control of disinfection and bather load strategies.
• Miniaturized or field-deployable enzymatic biosensors could allow on-site screening of urea and ammonia with rapid feedback to operators.
• Improved sample handling protocols and closed-system ammonia measurement can reduce atmospheric contamination and improve low-level precision.
• Data integration with laboratory information management systems (LIMS) and predictive maintenance for pool management (forecasting chloramine formation risk) are natural applications.

Conclusion

The Thermo Scientific enzymatic urea method (urease + GLDH) implemented on discrete analyzers provides a specific, automatable, and more accurate approach to urea monitoring in swimming pool water compared with the classical Koroleff persulphate digestion method. The enzymatic assays correlated closely with an independent enzymatic reference, achieved low detection limits (~0.064 mg/L), and performed well in proficiency testing. Main limitations arise from the calculated nature of the test (dependence on separate ammonia measurement) and increased relative error at low concentrations, which calls for careful handling and optimized protocols when measuring near regulatory limits.

References

1. Wojtowicz J. Cyanuric and Isocyanuric Acids. In: Kirk-Othmer Encyclopedia of Chemical Technology. John Wiley & Sons; 2000.
2. Koroleff F. Determination of Urea. In: Methods of Seawater Analysis. Grasshoff K, Erhardt M, Kremling K, editors. Weinheim: Verlag Chemie; 1983. p. 158–162.
3. Finnish Environment Institute (SYKE). Swimming Pool Water Report. 2013.