Introduction Scope of the review 1 Methodology is available for PCBs and OCPs as a result of a vast amount of environmental analytical chemistry research and development over the past 30 to 40 years. However, the establishment of an analytical laboratory and the application of this methodology at currently acceptable international standards is a relatively expensive undertaking. Furthermore, the current trend to use isotope-labeled analytical standards and high-resolution mass spectrometry for routine POPs analysis is particularly expensive. These costs limit participation of scientists in developing countries and this is clear from the relative lack of publications and information on POPs from countries in Africa, south Asia and South/Central America. Thus, this review will summarize the best practices in developed countries and new advances in PCB/OCP analysis, while bearing in mind the need for low-cost methods easily implemented in developing countries. 2 Substances to be analyzed no 3 4 5 1 6 7 6 8 cis  trans  cis  trans 9 10 11 12 13 no 14 2 143 144 Analytical standards 15 16 17 18 19 19 Commonly used analytical methods for PCB/OCP monitoring and surveillance Overview http://www.nemi.gov http://www.env.go.jp/en/index.html 20 21 22 23 http://www.ospar.org http://www.helcom.fi http://www.iso.org http://www.aoac.org http://www.krohne.ru/russia_en/downloads/certificates/cis/russia/gosstandard/ 24 25 26 27 28 Sampling 29 30 22 20 23 31 25 Blood samples should be collected in ethylenediamine tetraacetic acid (EDTA) vials or vacutainers, centrifuged, and the plasma transferred to vials washed with hexane. Field blanks, consisting of sample containers taken to the lab and returned with other samples, should be included. 3 3 3 32 33 34 35 36 37 38 39 40 41 41 42 43 44 45 45 46 47 48 49 44 47 Sample storage and handling 25 22 50 51 52 Sample preparation 53 54 25 55 56 30 57 1 2 Table 1 64 25 Technique Overview Method reference Conventional Soxhlet Sample + desiccant mixture in glass or paper thimble is leached with warm (condensed) solvent for 4–12 hrs. Solvents are, e.g., diethyl ether, DCM, hexane 65 Automated Soxhlet (e.g., “Soxtec”) Extraction thimble is immersed in boiling solvent (30–60 min) then raised for Soxhlet extraction. Solvent can also be evaporated. 65 Supercritical fluid extraction (SFE) Sample (usually +desiccant) placed in high-pressure cartridge and carbon dioxide at 150–450 atm at temp of 40–150 °C passed through. After depressurization, analytes are collected in solvent trap EPA 3560–3562 High-speed blending Useful for high water content samples such as plant material. Homogenizes sample with acetone and NaCl. 21 66 Column extraction Sample (+desiccant) placed in large column with filter and stopcock. Eluted with large volume of extraction solvent, e.g., hexane:DCM; hexane 67 Sonication-assisted extraction Sample in open or closed vessel immersed in solvent and heated with ultrasonic radiation using ultrasonic bath or probe. 65 Microwave-assisted extraction (MAE) Sample in open or closed vessel immersed in solvent and heated with microwave energy. 65 Pressurized liquid extraction (PLE) Sample (usually +desiccant) placed in extraction cartridge and solvent (heated, pressurized) passed through then dispensed in extraction vial. 61 65 Table 2 Guidance for various preparation, extraction and isolation steps in the analysis of PCBs and OCPs Environmental matrix Analytical steps General procedures EPA or other method Soil and sediment Preparation Prepare in a PCB- and pesticide-free room. 35 86 Avoid air-drying. Wet sieve if necessary to remove large particles. Centrifuge sediment to remove excess water. 2 4 Separate determination of dry mass by oven drying. For sediments total organic carbon should be determined. QA One blank, soil CRM every ten samples; spike all samples with recovery surrogate standards. Bake glassware overnight at 200 °C or higher. Extraction Soxhlet, PLE, sonication, or MAE with acetone: hexane or DCM Solvent evaporation, transfer to hexane. Sulfur removal with activated copper turnings required for sediment. Isolation/cleanup Alumina, silica or Florisil elutions: non-polar (hexane) and polar (DCM:hexane or equivalent) Vegetation Preparation Homogenize using food chopper or blender. Cryoblending using liquid nitrogen or dry ice is useful. Mix with dessicant. Separate determination of dry mass by oven-drying. 21 35 QA Same as soil. Use vegetation CRM if possible Extraction Same as soil. Isolation/cleanup Same as soil. Aquatic biota Preparation Select muscle or liver depending on species. For mussels and crustaceans use soft tissue. Select tissue that has not been in contact with the sample container. Homogenize using food chopper or blender. Cryoblending is useful. 21 35 Mix with drying agent. Separate determination of lipid content. QA Same as soil. Use fish or mussel SRMs. Extraction Soxhlet, pressurized liquid extraction, or column extraction. Use acetone:hexane or DCM. Isolation/cleanup Remove lipid using gel permeation chromatography if possible or by repeated washing of the extract with sulfuric acid. Follow with fractionation on silica or Florisil columns as described for soil. Marine mammal blubber Preparation Select blubber that has not been in contact with the sample container. Blend or hand-mix with drying agent. Separate determination of lipid content. 10 87  QA Same as soil. Use fish oil or marine mammal SRMs. Isolation/cleanup Same as for fish extracts. Air (high volume) Extraction,QA and cleanup Assuming that air is collected on polyurethane foams or XAD resin, these would be extracted in a Soxhlet or pressurized liquid extractor. Other steps as for soil or sediments 32 Semi-permeable membrane devices (SPMD) Preparation SPMDs would be removed from their transport cases and rinsed with precleaned water to remove accumulated dust (air-borne samplers) or periphyton (water samplers). 46 QA Use PRCs Extraction, and cleanup Assuming that the SPMD is lipid-based, extraction of POPs by “dialysis” into hexane would be achieved in a large glass cylinder. Water (including melted snow, ice and wet precipitation) Extraction Liquid–liquid, SPE (e.g., C18) extraction for small (<1 L) samples; XAD-2 or modified “Speedisk” for >1 L. 34 36 37 78 88 QA and cleanup Pre-spike XAD columns with surrogates. Blood plasma Extraction Extract blood plasma with ammonium sulfate/ ethanol/hexane (1:1:3) or C18 SPE extraction. 72 73 Determine lipid content. QA Same as fish. Use NIST 1589a SRM. Isolation and cleanup Sulfuric acid partitioning to remove lipids. Acid–base silica for additional lipid removal. Milk Extraction Liquid–liquid partitioning with acetone:hexane or C18 SPE extraction. Determine lipid content. 75 77 QA BCR SRM 284 & 533 milk powder. Isolation and cleanup As with plasma. Recommended extraction and isolation techniques for PCBs and OCP Recovery surrogates/internal standards 13 13 Extraction techniques 1 58 60 61 59 62 63 68 58 64 69 70 1 25 71 72 73 74 75 76 77 t 78 79 80 Determination of lipid content 25 81 82 83 84 85 Isolation of analytes from coextractives Bioanalytical methods for the quantification of OCPs and PCBs Adsorption “cleanup” columns 2  p p 89  p p 51 90 p p 21 76 26 Size-exclusion columns 67 91 Lipid destruction 92 93 87 Sulfur removal 94 95 Evaporation steps 96 21 21 Preparation for GC analysis Following fractionation on silica or Florisil, final extracts are prepared in GC vials for analysis. Addition of an internal standard to check solvent volume is recommended at this stage. Careful evaporation is required at this step, and only high-purity compressed gas (usually nitrogen) should be used. This can be done using a stream of regulated gas via a disposable glass pipet and heating block or via multineedle devices (e.g., “N-Evap”). Quantification methods Overview 4 97 Bioanalytical methods based on immunoassays, or in vitro bioassays for dioxin-like activity, have become widely available over the past ten years for the screening of sample extracts for POPs. These methods and selected applications are briefly reviewed in this section. GC injection ports 98 99 99 GC columns 99 100  n  n 18 101 3 2  n 18 101 2 Table 3 97 a Coeluting PCBs Number of chlorines A 4, 10 1, 2 A 9, 7 2, 2 A 12, 13 2, 2 A 17, 15 2, 3  A 27, 24 3, 3 A 32, 16 3, 3 A 28, 31 3, 3 A 33, 20, 53 3, 3, 4 A 43, 49 4, 4 A 47, 75, 48 4, 4, 4 A 44, 59 4, 4 A 37, 42 4, 4 A 71, 41, 64 4, 4, 4 A 66, 95 4, 5 A 56, 60 4, 4 A 84, 89, 101, 90 5, 5, 5, 5 A 117, 87, 115 5, 5, 5 A 77, 110 4, 5 A 135, 144, 124 6, 6, 5 A 147, 109 6, 5 A 123, 139, 149, 118 5, 5, 6, 5 A 114, 133 5, 6 A 131, 122 6, 5 A 153, 132, 105 6, 6, 5 A 176, 130 7, 6 A 164, 163, 138 6, 6, 6 A 158, 129 6, 6 A 175, 166 7, 6 A 173, 157, 201 7, 6, 8 A 170, 190 7, 7 A 198, 199 8, 8 A 203, 196 8, 8 a  p p  cis  trans 10  p p 102 Chiral GC separation of OCPs and PCBs  o p  cis trans 103 104 105 106 107 GC–ECD 4 14 63 2 2 108 Table 4 General guidance on GC analysis and data reporting for PCBs and OCPs GC detector Analytes Configuration Advantages/disadvantages a Capillary GC - with electron capture detection All ortho-subsituted PCBs & all OCPs on the POPs list except toxaphene 2 Relatively inexpensive and easy to operate. Similar response factors for most OCs. DDT/DDE ∼ 1 pg Good sensitivity for all POPs. Adequate for routine tasks. High potential for misidentification of some POPs due to coeluting peaks. HCB ∼0.5 pg Quadrupole mass spectrometry in electron ionization (EI) mode All PCBs & all OCPs on the POPs list except toxaphene 30 m×0.25 mm i.d. low-bleed columns with He carrier gas. Selected ion mode for target POPs.  Moderately expensive and more complex to operate and maintain. Newer instruments (post 1997) have adequate sensitivity for routine POPs monitoring at low pg/μL concentrations. Much less potential for misidentification than with ECD. DDT/DDE ∼1–10 pg HCB ∼1–10 pg Dieldrin ∼25 pg Toxaphene ∼500 pg (as tech mixture) Quadrupole mass spectrometry in electron capture negative ionization (ECNIMS) mode Toxaphene and other highly chlorinated OCPs and PCB with >4 chlorines 30 m×0.25 mm i.d. low-bleed columns with He carrier gas. Selected ion mode for target POPs. Comparable sensitivity in ECNIMS mode to ECD in SIM mode for some POPs. Much less potential for misidentification than with ECD. DDT/DDE ∼0.1 pg HCB ∼0.1 pg Dieldrin ∼1 pg Toxaphene ∼10 pg (as tech mixture) Ion trap mass spectrometry using MS/MS mode All PCBs, All OCPs on the POPs list 30 m×0.25 mm i.d. low-bleed columns with He carrier gas. Same columns as quadrupole MS. Comparable sensitivity to ECD in MS/MS mode for some POPs. Much less potential for misidentification than with ECD. DDT/DDE ∼1 pg HCB ∼1 pg Dieldrin ∼5 pg Toxaphene ∼100 pg (as tech mixture) High-resolution magnetic sector mass spectrometry in electron ionization (EI) mode All PCBs, all OCPs on the POPs list except toxaphene 30 m×0.25 mm i.d. low-bleed columns with He carrier gas. Selected ion mode for target POPs at 10,000× resolution. Comparable sensitivity to ECD in SIM mode. Highly reliable identification at low pg/μL levels. DDT/DDE ∼0.05 pg HCB ∼0.05 pg Dieldrin ∼0.1–0.5 pg Toxaphene ∼10 pg (as tech mixture) a GC–MS 109 110 90 35 111 112 13 Bioanalytical methods for the quantification of OCPs and PCBs Enzyme-linked immunoabsorbent assays (ELISA) 113 114 115 114 116  b 117 1 Fig. 1 Illustration of the basic components of an ELISA for detection of OCPs and PCBs in environmental samples or extracts. Sample antigen (analyte) competes with antigen for binding sites on coating protein; after a wash step, detection is performed by adding substrate and chromophore  118 119 120 Quality assurance issues for PCBs and OCPs Ancillary data 2 6 5 Table 5 Minimum reporting dataset for POPs analysis Information Details Sampling protocols Method, number, size and representativeness Storage temperature and location  Sample tracking information Date received, date analyzed, lab batch number or other unique identified Published analytical method e.g., EPA method Limit of detection/quantification QA procedures Blanks Reagents and also field blanks if possible Recoveries  Duplicates  Calibration Source of standards; date stocks prepared Surrogate and internal standards  QA of cofactors Such as lipid, organic carbon and moisture content Confirmatory tests e.g., Use of second GC column or other detection system Data manipulations Blank subtraction, recovery correction Field and lab blanks 35 QA procedures 121 25 74 122 As a routine measure, spiking surrogate recovery standards into each sample provides useful information on losses of analyte from the extraction step onwards. However, no single PCB or OCP can be representative of all of the organochlorines being determined, and thus recovery correction should be performed with caution. Isotopically labeled surrogates are ideal for analyses of PCBs and OCPs that are being performed by LRMS and HRMS, and isotope dilution techniques correct for the recoveries of these surrogates. 29 σ b Detection limits 3 145 152 122 6 122 σ  t  n Table 6 Detection limits defined by various organizations Organization Terminology Calculation US EPA Method detection limit (MDL) \documentclass[12pt]{minimal}
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\begin{document}$${\text{MDL}} = t_{{n - 1,\;99}} \times \sigma .$$\end{document} Minimum level of quantitation (ML) \documentclass[12pt]{minimal}
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\begin{document}$${\text{ML}} = 3.18 \times {\left( {t_{{n - 1,\;99}} \times \sigma } \right)}$$\end{document} American Chemical Society Limit of detection (LOD) \documentclass[12pt]{minimal}
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\begin{document}$${\text{LOQ}} = b + 10\sigma _{{\text{b}}} $$\end{document} International Organization for Standardization / International Union of Pure and Applied Chemistry (ISO/IUPAC) Critical value (CRV) minimum detectable value (MDV) \documentclass[12pt]{minimal}
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\begin{document}$${\text{CRV}} = t_{{n - 1,\;99}} \times \sigma _{{\text{b}}} $$\end{document} ISO/IUPAC \documentclass[12pt]{minimal}
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\begin{document}$${\text{LOQ}}_{{{{\text{ISO}}} \mathord{\left/ {\vphantom {{{\text{ISO}}} {{\text{IUPAC}}}}} \right. \kern-\nulldelimiterspace} {{\text{IUPAC}}}}} = K_{{\text{Q}}} \times \sigma _{{\text{Q}}} $$\end{document} 123  t n σ b  t  σ 124 126  b σ b \documentclass[12pt]{minimal}
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\begin{document}$${\text{LOD}} = b + 3 \times s_{{\text{b}}} $$\end{document}  b 122 n  n 122  b σ b 123 \documentclass[12pt]{minimal}
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\begin{document}$${\text{LOQ}}_{{{{\text{ISO}}} \mathord{\left/ {\vphantom {{{\text{ISO}}} {{\text{IUPAC}}}}} \right. \kern-\nulldelimiterspace} {{\text{IUPAC}}}}}  = K_{{\text{Q}}}  \times \sigma _{{\text{Q}}} $$\end{document}  σ Q  K Q 7 Table 7 127 Analyte ELISA (soil/fly ash) a b c PCB 28 – 0.05 0.1 0.01 PCB 52 – 0.05 0.1 0.01 PCB153 – 0.05 0.05 0.005 PCB180 – 0.05 0.02 0.005 p p 50 0.05 0.05 0.01 Toxaphene 500 0.05 0.02 0.005 Lindane 400 0.01 0.04 0.01 HCB – 0.01 0.02 0.005 Dieldrin 100–500 0.01 0.02 0.01 cis 100–500 0.03 0.05 0.01 Total PCB  100–500 0.1–1.0 0.1–1.0 0.01–0.1 a b 13 c 13 Reference materials 128 130 8 131 Table 8 128 CRM Source Tissue/species cis trans Dieldrin DDT HCB Mirex PCB congeners SRM1974b NIST mussel X X  X   X SRM1976 NIST lake trout X X  X   X SRM1588a NIST cod liver X  X X X  X SRM1945 NIST whale bl X X  X X X X SRM2974 NIST mussel X   X   X SRM2977 NIST mussel X X X X   X SRM2978 NIST mussel X  X X   X 140/OC IAEA plant   X X   X BCR598 BCR cod liver X X X X X  X CARP-1 NRCC carp       X BCR349 BCR cod liver       X BCR350 BCR mackerel       X BCR682 BCR mussel       X BCR618 BCR herring       X EDF 2525 CIL lake trout       X EDF 2514 CIL soil       X SRM1944 NIST sediment X   X X  X SRM1939a NIST sediment X   X   X IAEA383 IAEA sediment       X IAEA408 IAEA sediment    X X  X HS-1 NRCC sediment       X HS-2 NRCC sediment       X BCR536 BCR sediment       X DX-1 BCR sediment        DX-1 BCR sediment        Criteria for evaluating the desirability and efficacy of different analytical methods in environmental monitoring and surveillance Overview 3 145 152 7 3 132 Interlaboratory comparisons 27 53 133 134 26 PCB/OCP method accuracy 121 135 \documentclass[12pt]{minimal}
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\begin{document}$${\text{range}} = {\left( {\mu - {\left| b \right|} - 1.645\sigma } \right)}\;{\text{ to }}\,{\left( { - \mu + {\left| b \right|} + 1.645\sigma } \right)}$$\end{document}  μ  σ t n  σ b  σ b μ \documentclass[12pt]{minimal}
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\begin{document}$$\% \,{\text{accuracy}} = 100{{\left( {{\left| b \right|} + 1.645\sigma } \right)}} \mathord{\left/ {\vphantom {{{\left( {{\left| b \right|} + 1.645\sigma } \right)}} \mu }} \right. \kern-\nulldelimiterspace} \mu $$\end{document} 27 136 −50% to +20% for analytes in the range of <1 μg/kg −30% to +10% for analytes >1 μg/kg to 10 μg/kg −20% to +10% for analytes >10 μg/kg. These percentages are also recommended acceptability guidelines for samples spiked with PCBs/OCPs where no CRM is available. Emerging issues in analytical methods and future directions 58 137 138 139 140 no mo 141 142 Conclusions 6 The analytical methodologies discussed here refer to an “analytical system” encompassing information on the collection and storage of samples, the procedures used to extract, isolate, concentrate, separate, identify, and quantify POPs residues in samples, as well as specific quality control and reporting criteria. All aspects of this system must be in operation for POPs to be analyzed and reported. The chemical analytical methodology for the determination of PCBs and OCPs is a mature area within environmental analytical chemistry as a result of research and development over the past 30–40 years. Basic steps in the quantification of OCPs and PCB congeners have hardly changed in the past 20 years. Analytical methods for the determination of OCP/PCBs in foods, soils, sediments, fish, birds, mammals (including human milk and blood) are available and could be implemented at relatively low cost in developing countries. However, access to modern capillary GC equipment with either electron capture or mass spectrometry (MS) detection to separate and quantify PCBs/OCPs is required in order to conduct the analysis and to take part in regional and international intercomparsons. In general, ELISAs are very useful tools for the rapid assessment of PCB/OCPs contamination, especially in areas of former heavy use. They are particularly well-suited to laboratories in developing countries which may have access to spectrophotometric equipment but not to GC instrumentation. Existing analytical methods for PCB/OCPs can determine over 100 individual components at low ng/g concentrations in many environmental media using high-resolution capillary GC–ECD. However, the number of certified values for OCP/PCB congeners in certified reference materials is more limited (approximately 23 PCB congeners and 15 OCPs in NIST 1588a cod liver). At a minimum, the OCP/PCBs for which there are certified values in readily available CRMs should be determined (approximately 38). With this number of analytes, the information would be useful for both regulatory actions as well as for source identification using multivariate analysis or other “fingerprinting” methods. Interlab comparisons of POPs analysis over the past ten years have shown that availability of accurate analytical standards is a fundamental requirement of an analytical program designed to quantify trace organic contaminants such as POPs. Agencies such as GEF and UNEP Chemicals should give top priority to ensuring that certified analytical standards are available to all labs on a continuing basis. Quality assurance programs are critically important for demonstrating the performance of analytical methods for POPs within a lab and between labs. QA requirements for PCBs/OCP analysis are well known and include the use of certified reference materials, field and laboratory blanks, the use of quality control charts to monitor long-term lab performance, participation in interlaboratory studies, and the use of guidelines for sampling and analysis. Determination of PCBs/OCPs requires the analysis of blank samples because of the ubiquitous nature of these contaminants. If blanks are significant (for example, averaging greater than 10% of the average level of total PCBs), then blank correction should be carried out. As a routine measure, spiking surrogate recovery standards into each sample provides useful information on losses of analyte from the extraction step onwards. However, no single PCB or OCP can be representative of all the organochlorines being determined, and thus recovery correction should be performed with caution. Isotopically labeled surrogates are ideal for the quantification of PCBs and many OCPs via LRMS and HRMS; the application of isotope dilution techniques can correct for the recoveries of these surrogates. Detection limits depend not only on the analytical method used but also on the sample size and QA considerations, e.g., on information available from blank or control samples and recovery studies. Detection limits should be calculated as described by US EPA or by IUPAC/ISO methodology. Comparison of detection limits for widely used instrumentation for POPs with action limits for POPs in food and tissue residue guidelines suggests that current GC–ECD and GC-MS analytical methodology for PCB/OCPs can meet and exceed these limits, in some cases by orders of magnitude. Some emerging new analytical techniques, such 2D-GC and “fast GC” using GC–ECD, may be well-suited for use in developing countries in the near future given their relatively low cost and their ability to provide high-resolution separations of OCP/PCBs. Procedures with low environmental impacts (microscale, low solvent use, etc.) may be particularly well-suited to developing countries where analytical budgets are small and product delivery times are lengthy. Thus, strategies must be considered that will allow improved techniques to be adopted by such labs. Electronic supplementary materials Below is the link to the electronic supplementary material.
 Table 1 (DOC 4 kb) Table 2 (DOC 13 kb) Table 3 (DOC 6 kb)