Nitrate in Wastewater

Reflectometric determination after reduction to nitrite and reaction with Griess reagent.

Introduction

Excess nitrate in wastewater can lead to contamination of groundwater and subsequent environmental problems including eutrophication— excessive growth of algae and plants— upsetting marine ecosystems.¹ In addition, human consumption of excess nitrate has been linked to cancer and a blood condition called methaemoglobinaemia.² As a result, monitoring nitrate levels is critical for both environmental and health reasons. Here we describe nitrate determination in wastewater using reflectometry and the Reflectoquant^® Nitrate Test Kit.

Experimental Method

Nitrate ions are reduced to nitrite ions by a reducing agent. In the presence of an acidic buffer, these nitrite ions react with an aromatic amine to form a diazonium salt, which in turn reacts with N-(1-naphthyl)-ethylenediamine to form a red-violet azo dye that is determined reflectometrically.

Reagents and Instruments

Test Kits

• Nitrate Test Method: reflectometric with test strips 5 – 225 mg/l NO₃- Reflectoquant^® (1.16971) or
• Nitrate Test Method: reflectometric with test strips 3 – 90 mg/l NO₃- Reflectoquant^®

Instruments

• Reflectometer RQflex^® 20 Reflectoquant^® (1.17246)*

*Note: This application note pertains to RQflex^® 20 and all discontinued instruments (RQflex^® 10, RQflex^® plus).

Optional semi-quantitative determination

• Nitrate Test Method: colorimetric with test strips 10 - 25 - 50 - 100 - 250 - 500 mg/l NO₃⁻ MQuant^® (1.10020)

Sample preparation

The sample can be analyzed in its original form.

Instrumental Analysis

• Press the reflectometer START key and simultaneously dip the test strip into the sample (15 - 30 °C) for ca. 2 seconds, ensuring that both reaction zones are immersed.
• Allow excess liquid to run off via the long edge of the strip on to an absorbent paper towel.
• Wait 60 seconds and then measure the strip in the reflectometer.

The value [mg/l] will be stored automatically. (Please refer to the RQflex^® operating instructions and the instruction for use of the Reflectoquant^® Nitrate Test.)

Note: If a semi-quantitative determination is sufficient there is the possibility to use MQuant^® test strips, Cat. No. 1.10020 10 - 25 - 50 - 100 - 250 - 500 mg/l NO₃⁻

Procedure

Samples containing more than 500 mg/l NO₃^- must be diluted with distilled water. The pH must be within the range 1 - 12. If the pH is lower than 1, buffer the sample with sodium acetate; if it is greater than 12, adjust to approx. 3 - 5 with tartaric acid.

• Immerse both reaction zones of the test strip in the sample (15 - 25 °C) for 1 sec.
• Shake off excess liquid from the strip and after 1 min determine with which color field on the label the color of the NO₃^- reaction zone coincides most exactly.
• If the NO₂^- alert zone changes color, see “Notes on the measurement” in the instruction for use.
• Read off the corresponding result in mg/l NO₃^- or NO₃-N.

Results

Table 1. Comparison with photometric method

Sample	Reflectoquant® [mg/l]	Photometry [mg/l]
1	21	19
2	56	54
3	47	38
4	14	14
5	12	10
6	15	14
7	20	18
8	18	17
9	14	15
10	18	18
11	12	12
12	20	21
13	18	21
14	64	66
15	37	40
16	6	7
17	3	3
18	19	16
19	24	21
20	28	30

References

1. Camargo JA, Alonso Á. 2006. Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: A global assessment. Environment International. 32(6):831-849. https://doi.org/10.1016/j.envint.2006.05.002

2. 2008. Authority EFS. Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission to perform a scientific risk assessment on nitrate in vegetables. The EFSA Journal. 689:1-79. [Internet].