1. Roberto MORABITO ENEA - PROT morabito@casaccia.enea.it FONTI DI ERRORE NELL’ANALISI DI SPECIAZIONE ISA 2002 Le Spettroscopie nel Controllo Ambientale Val Masino 29 aprile – 1 maggio 2002

2. IUPAC DEFINITIONS « Speciation » (Chemical) Species: Specific chemical form of an element, defined according to its molecular, complex, electronic or nuclear structure Speciation analysis: Measurement of the amount of one or several chemical individual species in a sample Speciation of an element: Distribution of chemical species of an element in a system

4. DETERMINATION OF CHEMICAL FORMS OF ELEMENTS Critical steps Sampling, preservation (representative sample) Pre-treatment, conditioning (storage, drying, homogenisation) Chemical treatment (extraction, derivatisation, purification, etc.) Separation (chromatography) Detection, calibration (detector, calibrants)

5. UNCERTAINITY OF ENVIRONMENTAL MEASUREMENTS U T = (U 2 Sampling + U 2 Storage + U 2 Treatment + U 2 Analysis + …) 1/2

6. SAMPLING STRATEGIES CHOICE OF SAMPLES COLLECTION OF SAMPLES NUMBER OF SAMPLES SPATIA L VARIABILITY TEMPORAL VARIABILITY Filtered water - Particulate matter Undisturbed surface sediments Biota Sampling devices Sampling vessels Representativeness of sampling point Representativeness of sampling area Microlayer Interface water-sediment Vertical stratification Enclosed and flushed areas Dockyard activities Tides - Seasons

7. Depth Typical Tributyltin concentration profile in the sediments Resuspension Reduced direct inputs Degassing

9. SAMPLE STORAGE Adsorption on the container walls Contamination from the container walls Stability SAMPLE TREATMENT Contamination Losses Changes in speciation Low extraction efficiency Low derivatization yields SAMPLE ANALYSIS Interferents Matrix effects Calibration Blanks Analytical staff expertise SOURCES OF ERRORS

10. WATER SAMPLES Temperature pH Type of containers BIOLOGICAL AND SEDIMENT SAMPLES Temperature Humidity Type of containers PARAMETERS CONTROLLING THE STABILITY OF CHEMICAL SPECIES DURING STORAGE

11. SELECTION OF METHODS FOR SPECIATION ANALYSIS Stabilisation / Storage (examples) « Preserver the original speciation » STABILISATION - Acidification (waters) - - Lyophilisation (biota, sediments, etc.) - - Oven-drying (sediments) - - Pasteurisation (sediments) - - Gamma-irradiation (biota, sediments,waters) - STORAGE - Storage at ambient temperature for MeHg - - Storage at 4°C for trimethyllead - - Freezing recommended for butyl- and phenyl-tins (biota, sediments) -

14. SELECTION OF METHODS FOR SPECIATION ANALYSIS Extraction (examples) ACIDS Acetic acid (butyltins) Sulphuric acid, distillation (MeHg) Nitric acid/micro-wave (MeHg) MIXTURE ACID/ORGANIC SOLVENT Hydrochloric acid / toluene (MeHg, butyltins) Hydrochloric acid / methanol (butyltins) Sulphuric acid / toluene (MeHg) ORGANIC SOLVENTS Toluene (MeHg) Methanol / hexane (butyltins) Tropolone / pentane (butyltins) OTHERS CO2 / methanol, supercritical fluid (MeHg) CO2 / tropolone, supercritical fluid (butyltins) Protease / lipase (butyltins) TMAH (butyltins, IAL, TAL))

15. COMPARISON OF 12 SELECTED EXTRACTION METHODS FOR THE DETERMINATION OF ORGANOTINS IN BIOLOGICAL SAMPLES SAME SAMPLE (BCR 477) SAME OPERATOR SAME ANALYTICAL STEPS AFTER EXTRACTION THE PERFORMANCE OF THE OVERALL METHOD CAN BE STRICTLY RELATED TO THE EFFICIENCY OF THE EXTRACTION METHOD APPLIED

18. SELECTION OF METHODS FOR SPECIATION ANALYSIS Derivatisation (examples) « Transformation of a given compound to enable a better separation » HYDRIDE GENERATION (NaBH 4 ) R n Sn (4-n)+ -------- R n SnH (4-n) + H 2 with n = 1,2,3 (R= methyl, ethyl or butyl) ETHYLATION (NaBEt 4 ) PROPYLATION (NaBPr 4 ) PHENYLATION (NaBPh 4 ) GRIGNARD REACTIONS - Methylation - Ethylation - Propylation - Butylation - Pentylation - Hexylation

20. SELECTION OF METHODS FOR SPECIATION ANALYSIS Separation (examples) « necessary step, considering that the determination of different chemical species, in general, cannot be performed with a sufficient selectivity (e.g. selective detection by AAS) » GAS CHROMATOGRAPHY - Packed column - - Capillary column - HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) OTHERS - Cryoegenic trapping (cold trap) - - Capillary electrophoresis - - Ion exchange microcolumns -

21. SELECTION OF METHODS FOR SPECIATION ANALYSIS Detection (examples) ELEMENT SPECIFIC - Atomic absorption spectrometry - (electrothermal, flame, quartz furnace) MULTI-ELEMENT - Inductively coupled plasma atomic emission spectrometry (ICP-AES) - - Inductively coupled plasma mass spectrometry (ICP-MS) - SPECIFIC OF ONE GIVEN SPECIES - Voltammetry - NON SPECIFIC - Flame ionisation detection (FID) - - Flame photometric detection (FPD) - - Electron capture detection (ECD) - CALIBRATION - Stoichiometry, purity, primary and internal standards, calibration method (e.g. standard additions)

27. Analitycal methods for the determination of mercury compounds in environmental samples SEPARATION AND DETECTION CHROMATOGRAPHIC NON-CHROMATOGRAPHIC GC Packed column Capillary column HPLC Selective liquid-liquid extraction Selective reduction by SnCl 2. CV-AAS ECD (only for GC separation) MIP-AES, ICP-AES CV-AAS ICP-MS (MS)

29. Analitycal methods used for the determination of mercury compounds in environmental samples EXTRACTION Acid digestion and extraction in toluene CLEAN UP Back extraction in aqueous cysteine solution DERIVATIZATION Aqueous derivatization with NaBPh 4 GC-MS DETERMINATION

30. GC-MS characterization of phenylic derivatives of mercury compounds and instrumental parameters MeHgPh EtHgPh PhHgPh

31. ORGANOTIN ANALYSIS BY: GC-FPD GC-MS

32. TPrT TBT DBT MBT DPhT MPhT TPhT Inorganic Sn

33. DETERMINATION OF CHEMICAL FORMS OF ELEMENTS Sources of errors Compromise: To preserve the original « speciation » while permitting the analysis Sampling: contamination, losses, alteration of speciation Storage: contamination, losses, stability of species Extraction: contamination, incomplete recovery, degradation of chemical species Derivatisation: inhibition, degradation of chemical species Separation: peak overlap, degradation Detection: interferences Calibration: stability of species in solution, purity

34. MEASUREMENT QUALITY CONTROL GLP SOP’s Staff Training Instrument Control Calibrants and Blank Control MEASUREMENT QUALITY ASSESSMENT Independent methods Spiking experiments Reference materials Interlaboratory studies Statistic analysis TOOLS FOR QUALITY ASSURANCE

35. EUROPEAN PROJECTS FOR SPECIATION ANALYSIS ORGANOTIN COMPOUNDS IN THE ENVIRONMENT (1988-2002) JUSTIFICATION Toxicity of tributyltin and triphenyltin to marine biota (fish larvae, molluscs) EVALUATION OF THE STATE-OF-THE-ART DEVELOPMENT OF NEW METHODS PRODUCTION OF CRMS PARTICIPANTS Co-ordinations by the University of Pau, the University of Plymouth, ENEA and the Free University of Amsterdam Collaborations with the Joint Research Centres (Ispra and Geel) and the CID-CSIC (Barcelona) 23 laboratories from 9 EU countries Two bursaries

36. INTERLABORATORY STUDIES Improvement schemes (method performance studies) The principle followed is similar to a method validation, i.e.analyses of increasingly complex matrices to detect possible errors at the different analytical steps. These schemes have a pedagogic goal and involve technical meetings in the frame of which participants may exchange their experience and discuss weak points of their method(s) as well as source(s) of error(s). S1 S2 M1 M2 Simple solutions (pure substances or mixtures of substances) Solutions simulating the matrix or extracts (spiked or not) « Matrix » reference material similar to the sample, spiked with a known amount of the analyte « Matrix » reference material with a composition close to the sample composition DETECTION DERIVATISATION CLEAN-UP SEPARATION EXTRACTION MINERALISATION OVERALL METHOD

37. Test material – TBT spiked sediment Coordination BCR Preparation JRC of Ispra (IT) Collection Lake Maggiore (IT) Drying Air drying then oven drying (120 °C) Sieving 90  m mesh size Sterilization Not performed Homogeneization mixer Storage Brown glass bottles +4 °C Humidity < 4% Certified species None Participation 19 laboratories from 7 Countries

39. RM 424 Coordination University of Plymouth (UK) Preparation University of Pau (F) and IRMM (B) Collection Sado estuary (PT) Drying Air drying then oven drying (80 °C) Sieving 75  m mesh size Sterilization 120 °C for 1h Homogeneization mixer Storage Brown glass bottles +4 °C Humidity < 3% Certified species None Certification 18 laboratories from 8 Countries

41. BCR 477 Coordination ENEA (IT) Preparation ENEA (IT) and JRC of Ispra (IT) Collection La Spezia harbour (Italy) Drying Freeze drying Sieving 125  m mesh size Sterilization Not performed Homogeneization Mixer (15 days) Storage Brown glass bottles –20 °C Humidity < 5% Certified species TBT, DBT, MBT Certification 16 laboratories from 7 Countries

43. BCR 477 - TBT, DBT AND MBT IN MUSSEL TISSUE

44. BCR 710 Coordination ENEA (IT) Preparation JRC of Ispra (IT) and CNRS (FR) Collection Arcachon Bay (France) Drying Freeze drying Sieving 125  m mesh size Sterilization Not performed Homogeneization Mixer (15 days) Storage Brown glass bottles –20 °C Humidity < 5% Certified species TBT, DBT, CH 3 Hg, AsBet Certification 23 laboratories from 11 Countries

45. BCR 710 – As, Hg and Sn species in oyster tissue CompoundCertified value Standard deviation UnitSet of accepted results TBT13325  g/kg 10 DBT8217  g/kg 9 AsBet335mg/kg8 CH 3 Hg1158  g/kg 12

46. EUROPEAN PROJECTS FOR SPECIATION ANALYSIS ORGANOTIN COMPOUNDS IN THE ENVIRONMENT (1988-2001) WORK PROGRAMME Two trials (solutions, spiked sediment): 1988-90 Two coastal sediment certifications: 1990-93 Certification of a mussel CRM: 1994-97 Certification of a freshwater sediment CRM: 1997-99 Certification of an oyster CRM: 1999-2001 RESULTS Detailed studies of derivatisation methods Systematic studies of extraction recoveries Development of methods (SFE, HPLC-ID-ICPMS) One non-certified (harbour) sediment (RM 424) Two sediment CRMs (BCR 462 - BCR 646) One mussel CRM (BCR 477) One oyster CRM (BCR 710) M TBT (sediment) in µg/kg GC-FPD GC-MS GC-MIP-AES HPLC-ICPMS 15.025.035.0

47. CRMs AVAILABLE FOR ORGANOTIN SPECIATION ANALYSIS SEDIMENTS –PACS 2(TBT and DBT, NRCC) –RM 424(TBT, BCR) –BCR 462R (TBT and DBT, BCR) –BCR 646 (butyl- and phenyl- tins, BCR) FISH –NIES 11 (TBT and TPhT, NIES) MUSSELS –BCR 477 (butyltins, BCR) OYSTERS –BCR 710 (TBT and DBT, BCR) (Mulspot Project at the certification stage)

48. CRMs AVAILABLE FOR METHYLMERCURY SPECIATION ANALYSIS SEDIMENTS –IAEA 356 –BCR 580 CRUSTACEANS –NRCC / TORT-2 –NRCC / LUTS-1 OYSTERS –BCR 710 (Mulspot Project at the certification stage) ALGAE –IAEA 140 FISH –NRCC / DOLT-2 –NRCC / DORM-2 –BCR 463 –BCR 464 MUSSELS –NIST 1974a –NIST 2974 –NIST 2976 –NIST 2977

49. CRMs AVAILABLE FOR As, Cr, and Pb SPECIATION ANALYSIS MATERIALSPECIESPRODUCER/CAT. Dogfish muscleArsenobetaine, (CH 3 ) 4 As NRCC / DORM-2 Tuna fishArsenobetaine, DMA BCR 627 Oyster tissueArsenobetaineBCR 710 Freeze-dried waterCrIII, CrVIBCR 544 Urban dustTrimethylleadBCR 605

50. THE REGULATORY FRAME EC Directives Marine pollution, Conventions (North Sea, Mediterranean Sea, Rhine), Groundwater Directive Mention: « elements and their compounds » Consequences Chemical species are generally not determined by control laboratories Limited knowledge of their toxic impact and occurrence (studies needed) Analytical methods often at the prototype stage (still few commercialised instruments) Insufficient transfer of knowledge Needs Precise the chemical forms to be analysed (according to their toxicity) Make the techniques accessible to routine laboratories Develop multi-disciplinary synergies Educate legislators !