⚗️ Nitrate nitrogen in water and waste water — the colorimetric method with sodium salicylate (nitration in concentrated sulfuric acid, measurement at 410 nm)
In short
Nitrates react with sodium salicylate in the presence of concentrated sulfuric acid; nitrosalicylic acid is formed, which after being made alkaline passes into the dissociated form of an intensely yellow colour. The test is performed according to PN-C-04576-08:1982 (wersja polska); Range: ISO 7890-3: up to 0.2 mg N/l with a 25 ml sample; the range is extended upwards with a smaller volume of sample. The procedure comprises 8 steps (about 5 min in total); it is used for: Drinking water and raw waters — control of nitrates (a sanitary parameter, the risk of methaemoglobinaemia in infants), Municipal and industrial waste water — the nitrogen balance, control of the effectiveness of denitrification, Surface water and groundwater — monitoring of the inflow of nitrates from agriculture (the nitrates directive).
At a glance
- Standard: PN-C-04576-08:1982 (wersja polska)
- Category: Physicochemistry
- Procedure steps: 8
- Total time of stages: 5 min
- THE SAME DOCUMENT HAS A SECOND ARTICLE IN THIS DATABASE: PN-82/C-04576.08 is also described by a separate article on the same standard, marked as WITHDRAWN. That one gives the status of the document and its successor, this one describes the method itself. Both show the same list of laboratories, because they cite the same document
- Wavelength: 410 nm (PN-C-04576-08); 415 nm in ISO 7890-3, cells of 40 or 50 mm
- Range: ISO 7890-3: up to 0.2 mg N/l with a 25 ml sample; the range is extended upwards with a smaller volume of sample
Overview
The method is based on the reaction of nitration: nitrate ions in a medium of concentrated sulfuric acid nitrate sodium salicylate to nitrosalicylic acid, and this, after being made alkaline, gives a stable yellow colour measured spectrophotometrically. In a publication comparing methods of determining nitrates it is described thus: „Oznaczenia spektrofotometryczne wykonano według Polskiej Normy PN-82/C-04576/08 z zastosowaniem salicylanu sodu, który w obecności stężonego kwasu siarkowego reaguje z jonami NO₃⁻. W wyniku reakcji nitrowania powstaje kwas nitrosalicylowy, który po zalkalizowaniu przechodzi w postać zdysocjowaną o intensywnym żółtym zabarwieniu. Pomiary absorbancji wykonano przy długości fali 410 nm" [The spectrophotometric determinations were carried out according to the Polish Standard PN-82/C-04576/08 with the use of sodium salicylate, which in the presence of concentrated sulfuric acid reacts with NO₃⁻ ions. As a result of the nitration reaction nitrosalicylic acid is formed, which after being made alkaline passes into a dissociated form of an intense yellow colour. The absorbance measurements were carried out at a wavelength of 410 nm].
The same chemistry is described by the international standard ISO 7890-3:1988 (the method with sulfosalicylic acid), in which the sulfosalicylic acid is formed in situ by adding sodium salicylate and sulfuric acid to the sample. ISO gives the limits of the method: determination up to a nitrate nitrogen concentration of 0.2 mg/l with a 25 ml sample (the range is extended upwards with a smaller sample), a limit of detection of 0.003–0.013 mg/l in 40 mm cells, and a concentration of 0.2 mg N/l gives an absorbance of about 0.68 units. The method is intended for raw waters and drinking water; a colour of the sample itself of up to 150 mg/l Pt is permissible provided a correction of the absorption of the sample without the addition of the salicylate is carried out.
Interferences: the main interfering substances are chlorides, orthophosphates, magnesium and manganese(II). Nitrites are masked by the addition of sodium azide (or of sulfamic acid), and the precipitation of calcium and magnesium salts on making alkaline — by the addition of the disodium salt of EDTA together with the alkali. Samples of a pH above 8 are neutralised with acetic acid before the test portion is measured out; samples with suspended matter are left to stand, centrifuged or filtered through a washed glass fibre filter.
Status of the standard: PKN withdrew PN-C-04576-08:1982 (the earlier edition of 1973 concerned the method with phenoldisulfonic acid). The withdrawal does not invalidate the method in the laboratory — a withdrawn standard may be the basis of a testing procedure, but the scope of accreditation and the procedure must describe this clearly, and the laboratory should have a justification for continuing to use it. The regulation on the conditions for discharging waste water into waters or into the ground (Dz.U. 2019 item 1311) indicates for nitrate nitrogen molecular absorption spectrophotometry without assigning a definite standard and — as alternatives — flow analysis according to PN-EN ISO 13395 and ion chromatography according to PN-EN ISO 10304-1.
THE SCALE OF CITATIONS AND A CORRECTION OF THE KEY (23.09.2026). The field by which this page searches for laboratories has so far had the form "PN-C-04576-08" — i.e. the NEW scheme, with the year moved to the end. In the accreditation scopes, however, the OLD scheme predominates and the distribution is unambiguous: "PN-82/C-04576.08" 156 records, "PN-82/C-04576/08" 131, "PN-82/C-04576-08" 9, and the new "PN-C-04576-08:1982" — 9. The old key therefore showed 9 entries at 3 laboratories instead of 296 at 197. After the correction to "PN-82/C-04576" the tab covers all three sheet separators; it has been checked that the key does not pull in another sheet of the series — under the prefix PN-82 there stands ONLY sheet 08 (sheet 14 carries the year 1973, sheet 4 is written in the new scheme). The price of this change, stated plainly: nine records in the new scheme ("PN-C-04576-08:1982") fall outside the matching, because one prefix does not cover both generations of the record.
STATUS OF THE DOCUMENT — STANDARD WITHDRAWN. The PKN catalogue card gives: number "PN-C-04576-08:1982 - Polish version", date of publication 05-04-1982, DATE OF WITHDRAWAL 14-11-2012, "Supersedes: PN-C-04576-08:1973". The card carries the heading "Withdrawn" but has NO "Superseded by" row.
WHAT APPLIES TODAY FOR THIS TECHNIQUE. The answer is in a LEGAL ACT, not in the catalogue of standards. Annex No. 12 to the regulation of the Minister of Maritime Economy and Inland Navigation of 12.07.2019 (Dz.U. 2019 item 1311) lists three techniques for nitrate nitrogen: for "molecular absorption spectrophotometry (photocolorimetry)" — i.e. the technique this standard described — the "Standard" column contains a DASH, with no document; PN-EN ISO 13395 stands against flow analysis (CFA and FIA), and PN-EN ISO 10304-1:2009E against ion chromatography. PN-EN ISO 13395 is therefore NOT the successor of this standard — it stands against a DIFFERENT TECHNIQUE, and swapping the number would mean changing the way the test is performed.
Method principle
To the test portion a solution of sodium salicylate is added and it is evaporated to dryness in a water bath; after cooling, the dry residue is moistened with concentrated sulfuric acid, which causes the nitration of the salicylate by the nitrates present to nitrosalicylic acid. After dilution with water and making alkaline with a solution of sodium hydroxide with the addition of the disodium salt of EDTA, the dissociated form of an intense yellow colour is produced; the absorbance is measured at 410 nm (ISO 7890-3: 415 nm, cells of 40 or 50 mm) against a blank with water. The concentration is read off from a calibration curve prepared from potassium nitrate. According to ISO 7890-3 the reagents are: sulfuric acid c(H₂SO₄) = 18 mol/l (ρ = 1.84 g/ml), glacial acetic acid, an alkaline solution (200 g/l NaOH + 50 g/l disodium salt of EDTA dihydrate — prepared by carefully dissolving 200 ± 2 g of sodium hydroxide in about 800 ml of water, adding 50 ± 0.5 g of Na₂EDTA·2H₂O and making up to 1 litre; kept in a polyethylene bottle, stable indefinitely), a solution of sodium azide 0.5 g/l (0.05 ± 0.005 g in 100 ml of water — a very toxic reagent, which with acids evolves a highly toxic gas; alternatively sulfamic acid 0.75 g/l), a solution of sodium salicylate 10 g/l (1 ± 0.1 g in 100 ml of water, prepared daily before use), a stock standard nitrate solution of 1000 mg N/l (7.215 ± 0.001 g of potassium nitrate dried at 105 °C for at least 2 h, dissolved and made up to 1 litre; stable for up to 2 months) and working solutions of 100 mg N/l and 1 mg N/l (the latter prepared immediately before use).
Applications
- Drinking water and raw waters — control of nitrates (a sanitary parameter, the risk of methaemoglobinaemia in infants)
- Municipal and industrial waste water — the nitrogen balance, control of the effectiveness of denitrification
- Surface water and groundwater — monitoring of the inflow of nitrates from agriculture (the nitrates directive)
- Accredited laboratories declaring the method according to PN-C-04576-08 (a withdrawn standard, used as the basis of their own procedure)
- Serial analyses without a chromatograph — only a spectrophotometer and a water bath are needed
Key parameters
| Parameter | Value |
|---|---|
| THE SAME DOCUMENT HAS A SECOND ARTICLE IN THIS DATABASE | PN-82/C-04576.08 is also described by a separate article on the same standard, marked as WITHDRAWN. That one gives the status of the document and its successor, this one describes the method itself. Both show the same list of laboratories, because they cite the same document |
| Wavelength | 410 nm (PN-C-04576-08); 415 nm in ISO 7890-3, cells of 40 or 50 mm |
| Range | ISO 7890-3: up to 0.2 mg N/l with a 25 ml sample; the range is extended upwards with a smaller volume of sample |
| Limit of detection | 0.003–0.013 mg N/l (40 mm cells, 25 ml sample, ISO 7890-3) |
| Sensitivity | 0.2 mg N/l gives an absorbance of about 0.68 units (40 mm cells, 25 ml sample) |
| Main interferences | chlorides, orthophosphates, magnesium, manganese(II); nitrites — masked with sodium azide or sulfamic acid; calcium and magnesium — EDTA in the alkaline solution |
| Colour of the sample | permissible up to 150 mg/l Pt provided the absorption of the sample is corrected for (a determination without the salicylate) |
Standard
- Standard number
- PN-C-04576-08:1982 (wersja polska)
- Title (PL)
- Woda i ścieki — Badania zawartości związków azotu — Oznaczanie azotu azotanowego metodą kolorymetryczną z salicylanem sodowym
- Title (EN)
- Water and waste water — Tests for nitrogen compounds — Determination of nitrate nitrogen by colorimetric method with sodium salicylate
Step-by-step procedure
-
Sampling and preparation of the sample
Take the samples into glass bottles and analyse them as soon as possible after sampling; storage at 2–5 °C extends the stability of many kinds of samples, but this has to be checked for the given kind. Leave samples with suspended matter to stand, centrifuge them or filter them through a washed glass fibre filter. Neutralise samples of a pH above 8 with acetic acid.
-
Measuring out the test portion
Measure out into an evaporating dish a portion of a volume V such that it contains a mass of nitrate nitrogen within the range of the calibration curve (ISO 7890-3: at most 25 ml with a content of up to 0.2 mg N/l; at higher concentrations a smaller volume).
-
Removal of the interferences
Add the solution of sodium azide (or of sulfamic acid) so as to remove the influence of the nitrites; for samples with a high content of chlorides, orthophosphates, magnesium or manganese(II) dilute the sample or carry out the determination by the standard addition method.
-
Nitration
Add the solution of sodium salicylate, evaporate the contents of the evaporating dish to dryness on a boiling water bath, cool, moisten the dry residue with concentrated sulfuric acid and rub it with a glass rod until the residue dissolves; leave it to react.
-
Making alkaline and making up
Transfer quantitatively into a Nessler cylinder or a flask, add the alkaline solution with EDTA until a stable yellow colour is obtained, make up with water to the mark and mix. Equalise the temperature of the solutions (bath at 25 °C).
-
Measurement
Measure the absorbance at 410 nm (ISO 7890-3: 415 nm) against a blank prepared with water instead of the sample. For coloured samples carry out a second determination without the addition of sodium salicylate and subtract the absorbance measured.
-
Calibration curve
Prepare a series of standards from the working solution of potassium nitrate (prepared on the day of the determination) and put them through the full procedure. Determine the curve as the dependence of the absorbance on the concentration of nitrate nitrogen; check it with every series of determinations.
-
Calculation and recording
Read the concentration off from the calibration curve taking into account the volume of the test portion and the dilutions. Give the result in mg N-NO₃/l or mg NO₃/l — agree the unit with the client, because the difference of the conversion factor (4.43) is a frequent source of misunderstandings.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Spectrophotometer for measurements at 410–415 nm with cells of 40 or 50 mm | cells with a longer optical path raise the sensitivity | — |
| Evaporating dishes of a small capacity (about 50 ml) | new or rarely used ones are to be rinsed with water and put through the cleaning procedure before the first determination | — |
| Boiling water bath holding at least six evaporating dishes | evaporation of the test portion to dryness | — |
| Thermostatically controlled water bath at 25 °C ± 0.5 °C | equalisation of the temperature before the measurement | — |
| Nessler cylinders of 100 ml, volumetric flasks, one-mark pipettes | quantitative transfer and dilution | — |
| Glass fibre filters, centrifuge | removal of suspended matter before the determination | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Sodium salicylate, solution 10 g/l | 54-21-7 | 1 ± 0.1 g in 100 ml ± 1 ml of water; to be prepared daily before use, kept in a glass or polyethylene bottle |
| Concentrated sulfuric acid, c(H₂SO₄) = 18 mol/l, ρ = 1.84 g/ml | 7664-93-9 | the medium of the nitration reaction; eye protection and protective clothing are required |
| Alkaline solution: NaOH 200 g/l + Na₂EDTA·2H₂O 50 g/l | 1310-73-2 | making alkaline after the nitration; the EDTA prevents the precipitation of calcium and magnesium salts; stable indefinitely in a polyethylene bottle |
| Sodium azide, solution 0.5 g/l | 26628-22-8 | removes the interfering nitrites; very toxic, with acids it evolves a toxic gas — alternatively sulfamic acid 0.75 g/l |
| Glacial acetic acid, c(CH₃COOH) = 17 mol/l | 64-19-7 | neutralisation of samples of a pH above 8 before the test portion is measured out |
| Potassium nitrate (standard) | 7757-79-1 | 7.215 ± 0.001 g dried at 105 °C for ≥ 2 h per 1 litre = 1000 mg N/l; the solution is stable for up to 2 months in a glass bottle |
Health and safety (OHS)
- Concentrated sulfuric acid and glacial acetic acid — eye protection and protective clothing are obligatory, never pipette by mouth; add the acid to the water, not the other way round.
- Sodium azide is very toxic; contact of the solid reagent with acids releases a toxic gas. Do not introduce azides into drains with copper or lead pipes (the risk of the formation of explosive metal azides).
- The alkaline solution of 200 g/l NaOH is highly corrosive — prepare it in a cooled vessel, the dissolution is strongly exothermic.
- Carry out the evaporation to dryness under a fume hood; avoid overheating the residue after the addition of the acid.
- Collect waste with EDTA, azide and acids separately and hand it over for disposal in accordance with the regulations.
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-C-04576-08:1982 (wersja polska) — “Water and waste water — Tests for nitrogen compounds — Determination of nitrate nitrogen by colorimetric method with sodium salicylate”.
How does this method work?
To the test portion a solution of sodium salicylate is added and it is evaporated to dryness in a water bath; after cooling, the dry residue is moistened with concentrated sulfuric acid, which causes the nitration of the salicylate by the nitrates present to nitrosalicylic acid. After dilution with water and making alkaline with a solution of sodium hydroxide with the addition of the disodium salt of EDTA, the dissociated form of an intense yellow colour is produced; the absorbance is measured at 410 nm (ISO 7890-3: 415 nm, cells of 40 or 50 mm) against a blank with water.
What is the measuring range and accuracy?
THE SAME DOCUMENT HAS A SECOND ARTICLE IN THIS DATABASE: PN-82/C-04576.08 is also described by a separate article on the same standard, marked as WITHDRAWN. That one gives the status of the document and its successor, this one describes the method itself. Both show the same list of laboratories, because they cite the same document; Wavelength: 410 nm (PN-C-04576-08); 415 nm in ISO 7890-3, cells of 40 or 50 mm; Range: ISO 7890-3: up to 0.2 mg N/l with a 25 ml sample; the range is extended upwards with a smaller volume of sample; Limit of detection: 0.003–0.013 mg N/l (40 mm cells, 25 ml sample, ISO 7890-3).
How long does the test take?
The times given for the individual stages add up to approximately 5 min. The procedure comprises 8 steps.
What equipment is required?
Spectrophotometer for measurements at 410–415 nm with cells of 40 or 50 mm, Evaporating dishes of a small capacity (about 50 ml), Boiling water bath holding at least six evaporating dishes, Thermostatically controlled water bath at 25 °C ± 0.5 °C, Nessler cylinders of 100 ml, volumetric flasks, one-mark pipettes, Glass fibre filters, centrifuge.
Where is this test used?
Drinking water and raw waters — control of nitrates (a sanitary parameter, the risk of methaemoglobinaemia in infants); Municipal and industrial waste water — the nitrogen balance, control of the effectiveness of denitrification; Surface water and groundwater — monitoring of the inflow of nitrates from agriculture (the nitrates directive); Accredited laboratories declaring the method according to PN-C-04576-08 (a withdrawn standard, used as the basis of their own procedure); Serial analyses without a chromatograph — only a spectrophotometer and a water bath are needed.
What safety precautions apply?
Concentrated sulfuric acid and glacial acetic acid — eye protection and protective clothing are obligatory, never pipette by mouth; add the acid to the water, not the other way round.; Sodium azide is very toxic; contact of the solid reagent with acids releases a toxic gas. Do not introduce azides into drains with copper or lead pipes (the risk of the formation of explosive metal azides).; The alkaline solution of 200 g/l NaOH is highly corrosive — prepare it in a cooled vessel, the dissolution is strongly exothermic.; Carry out the evaporation to dryness under a fume hood; avoid overheating the residue after the addition of the acid.; Collect waste with EDTA, azide and acids separately and hand it over for disposal in accordance with the regulations.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-82/C-04576.