⚗️ Organochlorine insecticides (HCH, DDT, aldrin, dieldrin…), PCBs and chlorobenzenes in water — liquid-liquid extraction with hexane and GC-ECD

Physicochemistry PN-EN ISO 6468

Overview

ISO 6468:1996 describes a method for determining certain organochlorine insecticides, polychlorinated biphenyls (PCBs) and chlorobenzenes (excluding mono- and dichlorobenzenes) in drinking water, groundwater, surface water and wastewater; it is used for samples containing up to 0.05 g/l of suspended matter — in the presence of organic matter, suspended solids and colloids the interferences are more numerous and the limits of detection higher. The method gives information on individual PCB congeners, not on total PCBs. Limits of detection (table 1) for waters low in organic substances: organochlorine insecticides (α-, β-, γ-, δ-, ε-HCH, o,p'- and p,p'-DDE, -DDD, -DDT, methoxychlor, aldrin, dieldrin, endrin, heptachlor and its epoxide, α- and β-endosulfan) 1–10 ng/l depending on the compound; chlorobenzenes (tri-, tetra-, penta-, hexachlorobenzene, PCNB) 1–10 ng/l; PCBs 28, 52, 101, 138, 153, 180, 194 — 1–50 ng/l. At very low concentrations the problem of contamination is crucial — below 10 ng/l particular care is required. Every substance giving an ECD response at an indistinguishable retention time interferes; in practice many of them are removed by the extraction and the clean-up, and the use of two capillary columns of different polarity is usually sufficient (the results are treated as maximum concentrations). The 2019 regulation (Dz.U. 2019 poz. 1311 [Journal of Laws 2019, item 1311]) names PN-EN ISO 6468 as the GC reference method for aldrin, dieldrin, endrin, DDT, HCB, HCH, other insecticides, trichlorobenzene and PCBs in wastewater (for example a detection level of 1 µg/l for DDT, 0.5–1 µg/l for HCB). In Poland it is in force as PN-EN ISO 6468:2002 (Polish version).

In the PWr (Wrocław University of Science and Technology) textbook "Chemia wody" [Water chemistry] the determination of organochlorine pesticides is based on PN-EN ISO 6468:2002 (extraction with n-hexane — 1 h for samples without suspended solids, 4 h with them; distilling off to about 2 cm³, not to dryness; clean-up on Florisil; GC-ECD with a ⁶³Ni detector, a Zebron MultiResidue-1 column, injector and column at 195 °C, detector at 185 °C, argon 99.9 %; limits of determination γ-HCH 0.0005, DDE 0.002, DDD 0.003, DDT 0.005, methoxychlor 0.010 µg/dm³; the result is the mean of two determinations differing by no more than 15 %) and, as an alternative, on PN-EN 16693 (SPE with discs and GC-MS).

Method principle

Liquid-liquid extraction of the organochlorine insecticides, chlorobenzenes and PCBs with an extraction solvent; after the constituents of low volatility have been concentrated and, if necessary, cleaned up, the extracts are analysed by gas chromatography with an electron capture detector. Reagents: purified water (ion exchange or carbon), extraction solvent — hexane, petroleum ether or heptane (others if the recovery is at least 60 %; "pesticide grade" after being checked by concentrating 200 ml to 1 ml), anhydrous sodium sulfate (ignited for 4 h at 500 ± 20 °C), decane or dodecane as a "keeper", dry aluminium oxide (500 °C, 4 h) and deactivated aluminium oxide (7 % ± 0.2 % of water, 2 h), aluminium oxide/silver nitrate (0.75 g of AgNO₃ in 0.75 ml of water + 4.0 ml of acetone + 10 g of deactivated Al₂O₃; in the dark, to be used within 4 h), silica gel 63–200 µm (500 ± 30 °C for about 14 h; deactivated with 3 % of water, to be used within 24 h), toluene, peroxide-free diethyl ether, boiling granules washed with acetone, pure or certified standards (stock solutions e.g. 50 mg per 100 ml in acetone, pentane, hexane, dimethylbenzene or isooctane; stable for about 1 year at 4 °C in the dark), intermediate solutions (typically 10 µg/ml; 6 months) and working solutions (at least 5 concentrations in the ng/ml range; 1 month), a water-miscible solvent (acetone, methanol, DMF) for spiking. Apparatus: a GC with an ECD and capillary columns (glass or fused silica, internal diameter < 0.4 mm, 25–60 m; on-column injector or one with a glass liner; isothermal and programmed oven), separating funnels of 1–5 l with a glass stopcock washed with hexane or of PTFE, a high-speed stirrer and a magnetic stirring bar in PTFE, a micro-separator, a Kuderna-Danish apparatus with a Snyder micro-column, a rotary evaporator, a drying column (5–7 g of Na₂SO₄, a layer of 7–10 cm, e.g. 10 × 250 mm), clean-up columns (Al₂O₃/AgNO₃ 10 × 250 mm; silica gel — a macro-column 19 × 400 mm or a micro-column), microlitre syringes; glassware washed with detergent, with chromic or peroxodisulfate mixture, rinsed with hexane or heated for at least 12 h at 200 °C (not the calibrated glassware), with random blank checks. Samples: amber glass bottles of 1–5 l with a ground stopper or a PTFE-lined cap (not of plastics), filled to 80–90 %; pH 5–7.5 (adjusted immediately after sampling); for endosulfan a separate sample at pH 2; storage in the dark at about 4 °C, extraction as soon as possible (preferably within 24 h); dried extracts may be kept at 4 °C for up to two months and must not be evaporated to dryness. The volume of water is established by weighing the bottle before the extraction and after it has been emptied. Calibration: a) direct injections of the working solutions (1–10 µl) — linearity, retention times, relative responses; b) calibration of the whole procedure on spiked water samples (at least 5 levels, e.g. 1–200 ng/l, with less than 1 ml of solvent added per litre), from which the recovery of each analyte is calculated; daily recalibration; a blank of pure water through the whole procedure — a value above 10 % of the lowest result has to be explained.

Applications

• Drinking water and groundwater — residues of organochlorine insecticides (HCH/lindane, DDT and its metabolites, aldrin, dieldrin, endrin, heptachlor) at the ng/l level
• Surface water and wastewater — PCBs (congeners 28, 52, 101, 138, 153, 180, 194) and chlorobenzenes (HCB, PeCB, TeCB, TCB); reference method under Dz.U. 2019 poz. 1311
• Monitoring of priority substances and persistent organic pollutants (POPs) in the aquatic environment
• Samples with a low content of suspended solids (≤ 0.05 g/l) — trace analysis with control of contamination
• Laboratories with a GC-ECD and two capillary columns of different polarity; alternatively GC-MS after SPE (PN-EN 16693)

Key parameters

Standard

Standard number

PN-EN ISO 6468:2002 (ISO 6468:1996)

Title (PL)

Jakość wody — Oznaczanie wybranych chloroorganicznych insektycydów, polichlorowanych bifenyli i chlorobenzenów — Metoda chromatografii gazowej po ekstrakcji ciecz-ciecz

Title (EN)

Water quality — Determination of certain organochlorine insecticides, polychlorinated biphenyls and chlorobenzenes — Gas chromatographic method after liquid-liquid extraction

Step-by-step procedure

1. Sampling and storage

   Take the sample in accordance with ISO 5667-1/-2 into amber glass bottles of 1–5 l (not of plastics), filling them to 80–90 %; adjust the pH to 5–7.5 immediately after sampling (endosulfan — a separate sample at pH 2); keep in the dark at about 4 °C, away from concentrated solutions; extract as soon as possible (within 24 h). Establish the volume of water by weighing the bottle before and after emptying.

2. Extraction

   If the bottle is full, pour off 30–100 ml. Variant A: add 30 ml of the solvent, shake for at least 10 min, transfer to a separating funnel, return the aqueous phase to the bottle and repeat the extraction twice with 20–30 ml. Variant B: add 20–30 ml of the solvent, stir with a magnetic stirrer for at least 10 min at no less than 1000 rpm (or with a high-speed stirrer for 2 min at 4 °C) with the vessel covered, separate the phases in a micro-separator while adding purified water and take the extract with a pipette.

3. Drying

   Pass the extract through a column of anhydrous Na₂SO₄ that has been washed with the solvent (rinse it with a further 10–20 ml), or add Na₂SO₄ to the flask (shake for 1 min, leave for 5 min, decant, rinse with 10–20 ml), or freeze the extract at −18 °C for 2 h and decant it from above the ice (rinse with 10 ml).

4. Concentration

   Kuderna-Danish: with 2 boiling granules evaporate on a steam bath to 5 ± 1 ml, then with a Snyder micro-column or a gentle stream of nitrogen (bath ≤ 40 °C) to below 1 ml (below 0.5 ml add the keeper). Rotary evaporator: to at least 0.6 ml at a pressure not lower than 340 mbar, bath ≤ 50 °C; transfer quantitatively to a 1 ml flask and make up with the solvent. Do not evaporate to dryness.

5. GC-ECD and clean-up if required

   Analyse extracts of clean waters directly: inject 1–10 µl (as for the calibration) and compare the chromatogram with the standards. Where peaks overlap, clean up: on an Al₂O₃/AgNO₃ column (15 ml of solvent, 1.0 g of Al₂O₃/AgNO₃, 2.0 g of Al₂O₃, 5 mm of Na₂SO₄; apply the extract, rinse with 2 ml, elute with 30 ± 1 ml; if the whole column has turned black — repeat on a fresh one) or on silica gel (1 g in hexane, 0.2 g of Na₂SO₄; 100 µl of the extract; fractions of 2.5/2.5/8 ml of hexane, 8 ml of hexane/toluene 95:5, 16 ml of 90:10, 8 ml of hexane/ether 99.5:0.5; about 1–2 drops/s, nitrogen at about 25 mbar); concentrate the fractions and analyse them.

6. Calibration, blank and result

   Calibrate by direct injection of the working solutions (linear regression y = m·ρ + b; if the intercept is large, reject the highest concentrations) and by the whole procedure on spiked water samples (at least 5 levels, e.g. 1–200 ng/l; less than 1 ml of solvent per litre; on the day of use) — calculate the recovery; recalibrate every day. Run a blank through the whole procedure (above 10 % of the lowest result → look for the cause). Confirm the identification on the second column; report the result as a maximum concentration (co-elution is possible) in ng/l or µg/l.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Organochlorine insecticides and PCBs are toxic, persistent and bioaccumulative — gloves, fume hood, minimum quantities of standards, waste to be disposed of
• Hexane, petroleum ether, toluene and diethyl ether — flammable; ether forms peroxides (use peroxide-free ether)
• Chromic / peroxodisulfate mixture for washing the glassware — strongly corrosive and oxidising
• The ECD contains a ⁶³Ni source — handle it in accordance with the regulations on radioactive sources

Frequently asked questions