Sensitive Photometric Determination of ortho-Phosphate in Groundwater and Surface Water, Analogous to EPA 365.2+3, APHA 4500-P E, and DIN EN ISO 6878

Abstract

This protocol describes the sensitive determination of ortho-phosphate in groundwater and surface water using photometric analysis with ammonium molybdate. The procedure, based on EPA 365.2+3 and DIN EN ISO 6878 standards, utilizes Spectroquant^® test kits and UV/VIS spectrophotometers for accurate and efficient phosphate measurement. The method effectively detects low concentrations of PO₄-P, making it suitable for environmental water testing and monitoring.

Section Overview:

• Introduction
• Reagents, Instruments and Materials
• Experimental Procedure
• Analysis
• Results
• Summary
• References

Introduction

Phosphorus is an essential element for organisms and plants.¹ In natural, uncontaminated waters, it occurs as organically bound phosphate, condensed phosphates or as orthophosphate - often referred to by its chemical formula PO₄-P. The small quantity of phosphorus present in natural waters does not promote the growth of plants. However, a rise in the concentration of phosphorus results in the proliferation of algae, which leads to the eutrophication of the water body.^{2, 3}

In the mid-20^th century, the anthropogenic contamination of water bodies with phosphate has resulted in widespread eutrophication via fertilizers, wastewater, and washing detergents, among other things. Measures were taken to reduce phosphate concentrations through discontinuation of the use of phosphates in detergents and the precipitation of phosphates in wastewater treatment plants, which brought about a reduction in the phosphate environmental burden of approximately 75%.^{2, 4}

A prime example is the case of Lake Constance (Bodensee, Germany). At the end of the 1970s, the concentration of PO₄-P in the lake was 84 μg/L, whereas today this major water body exhibits PO₄-P levels of just 5-6 μg/L.^{5, 6}

Spectroquant^® Spectrophotometer Prove 600 plus

Analysis of phosphate in surface waters

The analysis of such low concentrations of PO₄-P is challenging. According to DIN EN ISO 10304-1, the lower working range of the ion chromatography method is 33 μg/L PO₄-P (equivalent to 100 μg/L PO₄^-), which is considerably higher than the phosphate concentrations usually present in surface waters.⁷ Photometry permits significantly more sensitive measurements. According to DIN EN ISO 6878, the lower measurement limit for the photometric determination of phosphorus in water is 5 μg/L PO₄-P, if no laborious extraction procedure is performed.⁸

Reagents, Instruments and Materials

Phosphate Test Kit/Reagents

For the measurement, one of the following Spectroquant^® test kits is necessary:

• Spectroquant^® Phosphate Test (1.14848)

Instrument(s) & Devices

For the measurement, one of the following Spectroquant^® photometers is necessary:

• Spectroquant^® UV/VIS Spectrophotometer Prove 600 Plus (1.73028)

Also, legacy systems

• Spectroquant^® Spectrophotometer Prove 600

Software for Data transfer

• Optional Spectroquant^® Prove Connect to LIMS software package (Y.11086) to transfer your data into an existing LIMS system.

Instrument Accessories

• Rectangular cells 100 mm (1.74011)

Other Reagents and Accessories

• Water for analysis (1.16754)
• Phosphate standard solution Certipur^® (1.19898)
• Preparation vessel, flat-bottomed long tubes with screw caps (1.14901)
• Plastic Syringes, 20 mL, Luer slip, polypropylene (XX1102012)
• Syringe filter holder, Millex-HPF LCR Filter, 0.20 μm, PTFE, with glass fiber prefilter (SLLGM25NS)
• Pipettes for 20 mL with plastic tips or 20-mL volumetric pipettes

Experimental Procedure

Sample Preparation

• Filter the sample as swiftly as possible after sampling over the above-mentioned filter into a clean and dry vessel.

Analysis

• Determine the PO₄-P content with the above mentioned test kit.
• For measurement in the 100-mm cell, both the sample volume as well as the quantities of reagents PO₄-1 and PO₄-2 must be quadrupled, i.e., using a sample volume of 20 mL, 20 drops of reagent PO₄-1 and 4 level microspoons of reagent PO₄-2.
• The measurement is carried out against a blank, prepared from water for analysis, and the reagents are prepared in an analogous manner.
• After measurement, the result can be read off of the instrument.

Results

As a means to gain expressive statements on the suitability of the Spectroquant^® test kit for the determination of the phosphate concentration in surface waters, six samples were investigated for their PO₄-P content using this test kit. For comparison, a reference analysis according to the DIN EN ISO 6878 method was also performed. A detection limit of 0.003 mg/L PO₄-P for the DIN method was determined in accordance with DIN (Table 1).

See more applications for photometry at Protocols and Application Notes

Table 1. Comparison of results of the Spectroquant^® Phosphate Test with a photometric reference analysis according to DIN EN ISO 6878

Groundwater and surface water	Concentration [mg/L PO4-P]
	Spectroquant® Phosphate Test 1.14848	DIN EN ISO 6878
Lake Starnberger See	< 0.0025	< 0.0030
Lake Wörthsee	< 0.0025	< 0.0030
Lake Altmühlsee	0.0126	0.0130
Lake Brombachsee	0.0025	0.0056
Lake Ammersee	< 0.0025	< 0.0030
Dam North Rhine-Westfalia	< 0.0025	< 0.0030

The two methods show comparable results: in four of the six samples, the PO₄-P concentration was below the measurement range. In only two of the six samples were the measured values within the range. In the sample from the Brombach Lake, the detected concentrations differed by 0.0031 mg/L PO₄-P. In the sample from Altmühl Lake, the deviation between the Spectroquant^® result and the DIN result was a mere 0.0004 mg/L PO₄-P. The recovery rate with regard to the DIN EN ISO method was 97%.

In addition to the reference analysis procedure, samples were also measured using the standard addition method. In this investigation, the five lake water samples and one reservoir sample were spiked with four different concentrations of PO₄-P and the recovery in each sample was determined. Table 2 shows the results of this investigation.

Table 2. Standard addition with the Spectroquant^® Phosphate Test

Groundwater and surface water	Addition [mg/L PO4-P]	Recovered concentration [mg/L PO4-P]	Recovery rate
Lake Starnberger See	0.0050	0.0055	110%
	0.0100	0.0101	101%
	0.0500	0.0516	103%
	0.1000	0.1005	101%
Lake Wörthsee	0.0050	0.0059	119%
	0.0100	0.0103	103%
	0.0500	0.0525	105%
	0.1000	0.1025	102%
Lake Altmühl	0.0050	0.0066	131%
	0.0100	0.0103	103%
	0.0500	0.0523	105%
	0.1000	0.1021	102%
Lake Brombachsee	0.0050	0.0069	137%
	0.0100	0.0108	108%
	0.0500	0.0520	104%
	0.1000	0.1027	103%
Lake Ammersee	0.0050	0.0061	123 %
	0.0100	0.0106	106 %
	0.0500	0.0524	105 %
	0.1000	0.1027	103 %
Dam North Rhine-Westfalia	0.0050	0.0059	119 %
	0.0100	0.0112	112 %
	0.0500	0.0532	106 %
	0.1000	0.1056	106 %

The added standard concentrations could be recovered in all samples. The average recovery rate was 105% ± 5%.

Note:

If the accuracy of the method is to be increased further, we recommend carrying out an own calibration for a limited measuring range. We recommend the performance of the calibration according to the ISO 8446-1 or respectively the DIN 38402-51 method.

For this purpose, a user-defined method can be programmed on the Prove 600 Plus.

For details, see Prove Manual chapter 9.6 “User-defined Concentration Methods”.

By recording a 10-point calibration curve for the measuring range 0.0005 - 0.0250 mg/L PO4-P, the following performance characteristics could be achieved:

Figure 1. 10-point calibration curve for the measuring range 0.0005 - 0.0250 mg/L PO₄-P

The method shows a good linearity. Compared with the pre-programmed method, the performance characteristics could be considerably improved.

Table 3. Performance characteristics

	Pre-programmed method 0.0025 – 0.5000 mg/L PO4-P	User calibration 0.0025 - 0.0250 mg/L PO4-P
Method standard deviation [mg/L]	+ 0.0029	+ 0.0001
Method coefficient of variation [%]	+ 1.2	+ 0.88
Confidence interval (P = 95 %) [mg/L]	+ 0.006	+ 0.0003

Summary

The Spectroquant^® Phosphate Test (1.14848) is a rapid, inexpensive, and precise alternative to the standard methods for the determination of orthophosphate in groundwater and surface waters. The results are comparable with those obtained using the DIN EN ISO 6878 standard method. By using the standard addition method, it was demonstrated that the test kit is suitable for the quantification of PO₄-P in groundwater and surface waters.

See more applications for photometry at Protocols and Application Notes

References

1. Belitz H, Grosch W. 1982. Wasser.1-6. https://doi.org/10.1007/978-3-662-08306-2_1

2. Gleisberg D. 1988. Phosphate und Umwelt. Chemie in nserer Zeit. 22(6):201-207. https://doi.org/10.1002/ciuz.19880220604

3. 1999. Standard Methods for the Examination of Water and Wastewater, 4500P – Phosphorus. 18. Washington, DC: American Public Health Association, American Water Works Association, Water Environment Federation.. [Internet].

4. Metzner G. 2006. Phosphate aus Wasch- und Reinigungsmitteln im kommunalen Abwasser der Bundesrepublik Deutschland. [Internet].

5. 2013. Internationale Gewässerschutzkommission für den Bodensee, Gesunder Bodensee dank niedriger Phosphorwerte. IGKB press release. [Internet].

6. Bodensee-Wasserversorgung, [cited 24 Aug 2016]. [Internet]. Available from: http://www.zvbwv.de/index.php?id=135

7. DIN EN ISO 10304-1:2009-07, Wasserbeschaffenheit_- Bestimmung von gelösten Anionen mittels Flüssigkeits-Ionenchromatographie_- Teil_1: Bestimmung von Bromid, Chlorid, Fluorid, Nitrat, Nitrit, Phosphat und Sulfat (ISO_10304-1:2007); Deutsche Fassung EN_ISO_10304-1:2009. https://doi.org/10.31030/1518948

8. DIN EN ISO 6878:2004-09, Wasserbeschaffenheit – Bestimmung von Phosphor – Photometrisches Verfahren mittels Ammoniummolybdat (ISO 6878:2004). [Internet].

9. 2016. Package leaflet Spectroquant® Phosphate Test. [Internet]. Available from: https://www.sigmaaldrich.com/catalog/product/mm/114848?lang=en&region=US