🌿 Aqua regia digestion of soil, treated biowaste, sewage sludge and waste before the determination of metals and other elements — heating under reflux (A1), in a heating block (A2) or by microwave in closed vessels at 175 °C (B), PN-EN ISO 54321

Environment PN-EN ISO 54321

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card of PN-EN ISO 54321:2021-07, in full (our translation of the Polish card text): “This document specifies two methods for digestion of soil, treated biowaste, sewage sludge and waste by the use of an aqua regia digestion. Digestion with aqua regia will not necessarily accomplish total decomposition of the sample. The extracted analyte concentrations may not necessarily reflect the total content in the sample but represent the aqua regia soluble metals under the conditions of this test procedure. It is generally agreed that for environmental analysis purposes the results are fit for the intended purpose — protection of the environment. This document is applicable to the following elements: aluminium (Al), antimony (Sb), arsenic (As), barium (Ba), beryllium (Be), boron (B), cadmium (Cd), calcium (Ca), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), magnesium (Mg), manganese (Mn), mercury (Hg), molybdenum (Mo), nickel (Ni), phosphorus (P), potassium (K), selenium (Se), silver (Ag), sodium (Na), strontium (Sr), sulfur (S), tellurium (Te), thallium (Tl), tin (Sn), titanium (Ti), vanadium (V) and zinc (Zn). This document can also be applied for the digestion of other elements, provided the user has verified the applicability.” We read the text of the standard in the preview of ISO 54321:2020 (first edition, 2020-08) from the cover to item b) of clause 9 (test report, page 7): introduction, clauses 1–8 in full.

ACCORDING TO THE TEXT OF THE STANDARD (introduction, clauses 1–8). The standard was prepared by ISO/TC 190/SC 3 together with CEN/TC 444 (Vienna Agreement). The introduction compares it with earlier documents: ISO 11466 requires a waiting time of 16 h after adding the acids before digestion, while EN 13657 and EN 16174 allow digestion to start once the first strong reactions have ceased; validation showed the difference between 2 h and 16 h of waiting to be negligible, so the standard follows EN 13657 and EN 16174; the heating block procedure was adopted from the Dutch NEN 6961 (boiling time 2 h to 4 h), here set at 2 h. Aqua regia digestion does not necessarily give complete element recoveries, and the extract may not reflect total concentrations; for most environmental applications the result is considered fit for purpose. Table 1 — validation matrices: sludges (municipal, industrial, from the electronic industry, ink waste sludge, sewage sludge), biowaste (method A only — compost, composted sludge), soil (agricultural, sludge-amended), waste (fly ash and bottom ash from municipal waste incineration, ink waste sludge, electronic industry sludge, reference materials BCR 146R and BCR 176). No normative references. Definitions: aqua regia — 1 volume of nitric acid (65 % to 70 %) and 3 volumes of hydrochloric acid (35 % to 37 %); dry residue — after drying at 105 °C ± 5 °C to constant mass. Principle (4): A1 — under reflux for (120 ± 10) min, A2 — in a heating block at (105 ± 5) °C for (120 + 10) min, B — microwave with temperature control, (175 ± 5) °C for (10 ± 1) min in a closed vessel; after each — filtration or centrifugation. Sources of errors (5): the sample container (elemental mercury penetrates polyethylene walls very fast in both directions, glass can introduce B, Na, K, Si and Al); grinding and milling (contamination from air, dust, wear of the mill; losses of volatile compounds — special care for Hg and As); filtration must not introduce contaminants; the whole test portion must be in contact with the acids; some elements can be lost by precipitation as poorly soluble chlorides, fluorides and sulfates. Reagents (6): analytical grade acids, reagent blank throughout; water (e.g. deionized), HCl ≈ 12 mol/l, HNO3 ≈ 15 mol/l, HNO3 ≈ 0,5 mol/l, antifoaming agent (e.g. n-dodecane or n-octanol). Apparatus (7): A1 — vessel (e.g. quartz; silica or borosilicate permitted) with a volume of at least 5 times the volume of aqua regia, reflux condenser, absorption vessel for volatile species, thermostatically controlled heater; A2 — 50 ml tube with a polypropylene screw cap (the unheated part acts as a condenser but is not a reflux system; the material must be tested for release of the elements of interest) and a heating block at (105 ± 5) °C; B — pressure vessel typically 100 ml of PFA, modified PTFE or quartz, withstanding at least 3 000 kPa, with a safety system; laboratory microwave system with temperature feedback: control ±5 °C, power adjustment within 2 s, sensors accurate to ±2 °C; the accuracy of the temperature measurement is checked periodically on a blank — with a deviation of more than 2 °C from an external calibrated thermometer the system must be recalibrated; filters usually 0,45 µm, flasks 50 ml or 100 ml, balance accurate to 1 mg or better, boiling aids 2 mm to 3 mm. Procedure (8): soil, sludge and biowaste samples are pre-treated e.g. according to EN 16179 or ISO 11464, waste — e.g. according to EN 15002; dry matter is determined e.g. according to EN 15934; solid waste is dried or reduced below 250 µm, liquid waste homogenized; the test portion must be representative and comply with the instrument manufacturer’s instructions — for representativeness more than 200 mg is preferred; reagent blank in parallel, its acceptance limits are set by the laboratory. A1: approx. 3 g (waste 1 g to 10 g) weighed to 0,001 g (or at least three significant figures); a dry sample is moistened with 0,5–1,0 ml water, (21,0 ± 0,1) ml HCl and (7,0 ± 0,1) ml HNO3 are added; approx. 15 ml of 0,5 mol/l HNO3 into the absorption vessel; left to stand until effervescence almost ceases and — for consistency — heating is recommended to start as soon as possible after the first strong reaction has ceased; 30 ml aqua regia is sufficient to oxidize approx. 0,5 g organic carbon — with more, 1 ml HNO3 is added per 0,1 g above 0,5 g, no more than 10 ml at a time; boiling under reflux 2 h, condensation zone below 1/3 of the condenser height; the content of the absorption vessel is poured through the condenser, rinsed with 10 ml of 0,5 mol/l HNO3, made up to the mark. A2: at most 2 g (typically 0,5 g to 1 g of dry sample) with at most 0,15 g organic carbon for 8 ml aqua regia — for each additional 0,1 g carbon, 1 ml concentrated HNO3 before digestion (the standard stresses that for e.g. barium and chromium this addition is essential for sufficient recovery); (6,0 + 0,1) ml HCl and (2,0 + 0,1) ml HNO3; cap loose; boiling temperature for (120 + 10) min. B: at most 2 g (typically 0,5 g to 1 g) with at most 0,15 g organic carbon, 1 ml HNO3 per additional 0,1 g carbon; (6 ± 0,1) ml HCl and (2 ± 0,1) ml HNO3; with a vigorous reaction wait before closing, with foaming — a drop of antifoaming agent; heating at approx. 10 °C/min to 15 °C/min to (175 ± 5) °C and (10 ± 1) min at that temperature — the method is operationally defined; too fast a temperature increase may cause a vigorous exothermic reaction, a pressure rise, opening of the safety valve and analyte losses; after cooling the vessels are checked for tightness. In every method adjustment to volume with the residue still present immediately after digestion is also allowed; a solution with particles may be centrifuged, allowed to settle or filtered; the solution is ready for the determination of elements by an appropriate technique. The test report (9) contains at least the digestion method used with reference to ISO 54321 and the identity of the sample (further items we do not have). The table of contents also lists informative Annexes A and B with repeatability and reproducibility data for soil, biowaste and sludge and for waste.

STATUS (cards as of 02.10.2026). The card of PN-EN ISO 54321:2021-07 (English version) has no “Withdrawn” header — the standard is CURRENT: published 02-07-2021, 52 pages, price group U, Health, Environment and Medicine Sector, KT 191 for Soil Chemistry, ICS 13.080.10, “Introduces: EN ISO 54321:2021 [IDT], ISO 54321:2020 [IDT]”; the card has no “Replaces” field. There is no Polish version in the catalogue: the PKN search for “PN-EN ISO 54321” (02.10.2026) shows one item. Documents with which the introduction compares it: card of PN-EN 16174:2012 (English version; sludge, treated biowaste and soil — aqua regia digestion, three methods) — “Withdrawn”, withdrawal date 30-03-2022, without a “Replaced by” field; card of PN-EN 13657:2006 (Polish version; characterization of waste — aqua regia digestion) — no “Withdrawn” header, i.e. still current in the PKN catalogue alongside PN-EN ISO 54321. We have not checked the status of ISO 54321:2020 on the ISO website. Legislation: the Regulation of the Minister of the Environment of 6 February 2015 on the use of municipal sewage sludge (consolidated text Dz.U. 2023 item 23) as amended by the Regulation of the Minister of Climate and Environment of 14 January 2022 (Dz.U. 2022 item 89), which in this part entered into force 48 months after publication (in the Sejm ELI database — 15.01.2026): Annex 4 for the content of heavy metals (lead, cadmium, mercury, nickel, zinc, copper and chromium) indicates “atomic absorption spectrometry after mineralization in aqua regia or concentrated acids, the determination error not exceeding 10% of the relevant limit value, or inductively coupled plasma optical emission spectrometry (ICP-OES), or inductively coupled plasma mass spectrometry (ICP-MS), or atomic absorption spectrometry with amalgamation (mercury only)” — it gives no standard number; ICP-MS and the amalgamation technique were added by the 2022 wording (the earlier one named only AAS after mineralization and ICP-OES). The same amendment (§ 2 para. 5) requires the tests to be carried out by an accredited laboratory or one with a certified quality management system covering this procedure; samples taken before the amendment entered into force may be tested under the previous rules.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, data up to 17.09.2026, read 02.10.2026). The number 54321 appears in 21 records of 8 laboratories: AB 1036 and AB 1044 (4 each), AB 127 and AB 176 (3 each), AB 216, AB 447, AB 697 (2 each), AB 1173 (1). Current scopes on the PCA website (read 02.10.2026, scope issues from 05.11.2025 to 03.08.2026): the number stands in 21 lines — the same as the records in the copy. Edition: everywhere 2021-07, written in different ways — “PN-EN ISO 54321: 2021-07” with a space (AB 697), “PN EN ISO 54321:2021” without hyphen and without month (AB 216, in one of two items), “PN-EN ISO 54321:2021-07 metoda B” (AB 176, in all three items); the other laboratories do not indicate the method (A1, A2, B). None of these items is suspended (at AB 176 the footnote on the suspension of part of the scope from 03.06.2025 concerns lines in bold italics — the lines with 54321 are not set that way). The “Laboratories” tab searches for entries BEGINNING WITH “PN-EN ISO 54321” and shows all 8 laboratories — checked with the tab query on the production server labcoda.pl on 02.10.2026: 8 (under “PN EN ISO 54321” — 1, the same AB 216; under “EN ISO 54321” and “ISO 54321” — 0).

WHAT THE LABORATORIES DETERMINE (items in PCA). Object: sewage sludge — at all 8 laboratories; waste with code 19 08 05 (stabilized municipal sewage sludge) — AB 127, AB 176, AB 447, AB 1036, AB 1044; waste with code 19 01 14 — AB 697; waste with code 17 05 04 — AB 176; soil — AB 127 and AB 176 (“soil, ground”). Under the heading of the regulated area of the regulation on the use of municipal sewage sludge (Dz.U. 2023 item 23) the items with this standard stand at AB 127 (waste 19 08 05) and AB 447. Determination technique and measurement standard alongside: ICP-OES — according to PN-EN ISO 22036:2024-07 (AB 127, AB 176, AB 1173) or PN-EN ISO 11885:2009 (AB 127 — soil, AB 216, AB 447); ICP-MS according to PN-EN ISO 17294-2:2024-04 — AB 697; FAAS according to PN-ISO 8288:2002 (AB 1036 with “Method A”, AB 1044) and PN-EN ISO 7980:2002 (AB 1044, calcium and magnesium); mercury — CVAAS according to PN-EN 16175-1:2017-02 (AB 216) or AAS with amalgamation according to PN-EN ISO 12846:2012 + Ap1:2016-07 (AB 1044), and also ICP-MS (AB 697, (0,10–50) mg/kg) and ICP-OES (AB 1173, (1,00–200) mg/kg); total phosphorus spectrophotometrically according to PN-EN ISO 6878:2006 with corrigenda Ap1 and Ap2 (AB 447 — point 7, AB 1036 — point 8, AB 1044). Ranges, e.g.: cadmium (0,200–20,0) mg/kg (AB 447), (0,50–1000) mg/kg (AB 176), (5,0–1000) mg/kg (AB 1036); zinc up to 10000 mg/kg (AB 216, AB 447, AB 697); calcium up to 200000 mg/kg (AB 697, AB 1044) or (1,00–50) % (AB 447); phosphorus up to 200000 mg/kg (AB 697).

NEXT TO THIS STANDARD. The measurement standards listed above — all have separate entries in the Knowledge Base except PN-EN 16175-1; no other digestion documents stand next to PN-EN ISO 54321 in these items. Status of the measurement standards (cards as of 02.10.2026): cards of PN-EN ISO 22036:2024-07, PN-EN ISO 11885:2009, PN-EN ISO 17294-2:2024-04, PN-EN 16175-1:2017-02, PN-EN ISO 12846:2012, PN-ISO 8288:2002, PN-EN ISO 7980:2002 and PN-EN ISO 6878:2006 (with additional elements Ap1:2010 and Ap2:2010) — all without a “Withdrawn” header.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. In preparation: a polyethylene container loses and takes up elemental mercury, glass introduces B, Na, K, Si and Al; grinding and drying risk contamination and loss of volatile compounds (Hg, As) — the result for mercury and arsenic depends on what happened to the sample before digestion. In digestion: too little nitric acid with a high content of organic carbon (A1 — more than 0,5 g, A2 and B — more than 0,15 g) lowers recovery, and for barium and chromium the standard calls the HNO3 addition essential; the standing time before heating affects the degree of digestion (the standard recommends starting right after the first strong reaction); in method B too fast heating can open the safety valve with loss of analytes, and a vessel that leaked during digestion must be detected; poorly soluble chlorides, fluorides and sulfates may precipitate part of the elements. In interpretation: the result is the fraction soluble in aqua regia under the conditions of the method, not the total content — methods A1, A2 and B are operationally defined, so a result without stating the method (as in the scopes of 7 of 8 laboratories) does not say which route the sample took; biowaste was validated with method A only. In the calculation: the content in dry matter combines the concentration in the solution (measured by a spectrometric technique according to another standard), the flask volume and the test portion mass (of this method) and the dry matter (determined according to another standard, e.g. EN 15934); the reagent blank is subtracted according to limits set by the laboratory. In legislation: the sludge regulation allows mineralization in aqua regia or concentrated acids and does not indicate method A1, A2 or B; in its wording the 10 % error limit of the limit value applies to atomic absorption spectrometry.

WHAT WE DO NOT GIVE. We do not have clause 9 beyond items a) and b) or Annexes A and B (repeatability and reproducibility data). We do not give the content of EN 13657, EN 16174, ISO 11466 or of the sample preparation standards (EN 16179, ISO 11464, EN 15002) and dry matter (EN 15934) — we name them as the text of the standard refers to them. We do not reproduce the limit values for metals in sludge from the regulation.

Method principle

A sample of soil, sludge, biowaste or waste is digested with aqua regia (1 volume HNO3 to 3 volumes HCl) — under reflux for 2 h (A1), in a heating block at boiling temperature for 2 h (A2) or by microwave in a closed vessel at (175 ± 5) °C for 10 min (B) — and the solution, after filtration or centrifugation and making up to volume, is analysed by a spectrometric technique according to another standard. The methods are operationally defined: the result is the fraction of elements soluble in aqua regia under the conditions of the method, not always the total content.

Applications

• Heavy metals, calcium, magnesium and phosphorus in sewage sludge and waste with code 19 08 05 — at all 8 laboratories, including under the heading of the regulated area of the regulation on the use of municipal sewage sludge (AB 127, AB 447)
• Elements in soil and ground (AB 127, AB 176) and in waste with codes 19 01 14 (AB 697) and 17 05 04 (AB 176)
• Mercury after aqua regia digestion — CVAAS (AB 216), AAS with amalgamation (AB 1044), ICP-MS (AB 697), ICP-OES (AB 1173)

Key parameters

Standard

Standard number

PN-EN ISO 54321:2021-07 (wersja angielska; EN ISO 54321:2021, ISO 54321:2020 IDT)

Title (PL)

Gleba, uzdatnione bioodpady, osady ściekowe oraz odpady — Roztwarzanie frakcji pierwiastków rozpuszczalnych w wodzie królewskiej

Title (EN)

Soil, treated biowaste, sludge and waste — Digestion of aqua regia soluble fractions of elements

Step-by-step procedure

1. Sample preparation

   Soil, sludge, biowaste — e.g. according to EN 16179 or ISO 11464; waste — e.g. according to EN 15002 (solids below 250 µm); dry matter e.g. according to EN 15934. PN-EN ISO 54321:2021-07 current (card as of 02.10.2026).

2. Test portion and acids

   A1: approx. 3 g (waste 1–10 g), 21 ml HCl + 7 ml HNO3; A2 and B: ≤ 2 g (typically 0,5–1 g of dry sample), 6 ml HCl + 2 ml HNO3; moisten a dry sample with water; with excess organic carbon — 1 ml HNO3 per 0,1 g above the limit.

3. Standing

   Until effervescence almost ceases; heating is recommended to start right after the first strong reaction has ceased. Reagent blank in parallel.

4. Digestion

   A1: boiling under reflux 2 h (condensation below 1/3 of the condenser height); A2: boiling in the block (120 + 10) min with the cap loose; B: 10–15 °C/min to (175 ± 5) °C, (10 ± 1) min, cooling, check of vessel tightness.

5. Solution for measurement

   A1: content of the absorption vessel through the condenser, rinsing with 10 ml of 0,5 mol/l HNO3; making up to the mark; particles — centrifugation, settling or filtration (0,45 µm).

6. Determination and report

   Elements by a spectrometric technique according to another standard (in the scopes: ICP-OES, ICP-MS, FAAS, CVAAS, AAS with amalgamation, spectrophotometry of phosphorus); the report contains at least the digestion method with reference to ISO 54321 and the identity of the sample.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Highly corrosive and very toxic acids, high temperature and pressure — work in a fume hood or in forced-ventilated equipment; do not open pressure vessels before they have cooled; with samples rich in organic matter explosive intermediates are possible
• Only a laboratory-grade microwave digestion system with corrosion-resistant safety devices — a domestic microwave oven must not be used (acid vapours may damage the magnetron interlock when the door is open)
• TECHNICAL NOTE: the result is the fraction soluble in aqua regia under the conditions of the chosen method (A1, A2, B), not always the total content — the method must be stated with the result

Frequently asked questions