209 PCBs understanding all polychlorinated biphenyls

209 PCBs understanding all polychlorinated biphenyls Everyone talks about 209 PCBs, but what is it and why is it important for us? And is it difficult to analyze? Frank Neugebauer Eurofins GfA Lab Service GmbH Neulaender Kamp 1, 21079 Hamburg, Germany 23.04.2015 Miljøgifter i fisk og sjømat - Alesund, Norway 23rd April 2015 www.eurofins.de

PCB and their significance 2

Polychlorinated Biphenyls (PCB) PCBs are a group of polychlorinated aromatic hydrocarbons 3 2 2 3 4 4 Cl x 5 6 6 5 Cl y x,y = 0...5; x+y>0 209 possible congeners Congener = substance having the same basic molecule but different number and position (distribution) of substituents 3

PCBs: synthesis, sources and usage Main source of PCB = industrial production! Synthesis: non-specific chlorination of biphenyl technical mixture Preferred formation of specific single PCB-Isomers typical distribution pattern for technical PCB mixtures Trade names: Arochlor (US), Chlophen (DE), Kanechlor (JP), Pyralen (FR) etc. Different usages, e.g. Insulation fluids (transformers, condensators) Hydraulic oils Drill oils Additives for building materials (e.g. sealings, glues, colorants) Lubricants 4

PCBs: further sources and heritage Industrial production For decades, since begin of the 1920s, in total over 1 million tons worldwide A major part of these PCBs are still not disposed of Another part has been accumulated in the ecosphere Secondary sources unintentional formation (by-products) Synthesis of chemicals, e.g. dyes and colorants: specific reactions from input chemicals distinct patterns Combustion processes (including combustion of technical PCBs) see also: dioxin formation! Distribution, e.g. between environmental compartments E.g. spilled oil water fish 5

The problem with the PCBs Only low water-, but high lipid solubility (lipophilic) Chemical, physical and biological stability global, ubiquituous distribution very long biological half lifes (high persistency) (e.g. ca. 2,7a in humans for PCB #126) accumulation in the food chain (especially dioxin-like congeners) accumulation in humans Partially highly toxic (animal experiments and observation of human exposition) 6

Global contamination paths of PCDD/F and dl-pcbs Bio-Akkumulation Deposition PCDD/F PCB PCDD/F PCB- Eintrag Emission Bio-Akkumulation Bio-Akkumulation 7

Toxicity 8

Toxic effects depending on single compounds Difficult assessment: complexity of mixtures and strongly different toxicity of single compounds Non-dioxin-like toxicity of PCB (ndl-pcb) moderately toxic, long-term toxicity; organ dysfunction (liver), immune system disruptions, skin alterations, non-specific symptoms Cases of human death (e.g. contaminated rice oil: Yusho 1968, Yu Cheng 1979) Further toxicity and limit value discussions since 2010s; adressing different modes of toxic action for dl-/ndl-pcb EU limit values Dioxin-like toxicity of PCB (dl-pcb) Toxicity discussion of dl-pcb since 1990s; TEF assignation (WHO-TEF) Highly toxic; presumably cancerogenic effects analog to 2,3,7,8- Tetrachlordibenzo-p-dioxin Long half-lives in human body: PCB 77 0,1a; PCB 126 2,7a; PCB 169 13a (Liem und Theelen, 1997) 9

PCBs: relevance of single congeners PCBs are a group of polychlorinated aromatic hydrocarbons 3 2 2 3 Cl Cl Cl Cl Cl x 4 5 6 6 5 4 Cl y Cl Cl Cl Cl 0,3nm x,y = 0...5; x+y>0 O 209 possible congeners 1nm 12 relevant dl-pcbs with dioxin-like properties, (0 or 1 chlorine substitution in positions 2,2,6,6 ) 6 relevant ndl-pcbs = non-dioxin-like properties = Marker/Indicator-PCBs (#28, #52, #101, #138, #153, #180 sometimes + #118 = 7) 10

Dioxin-like toxicity: TEF-Concept (Equivalent-factors acc. to WHO) highest toxicity (TEF human / mammals) lowest toxicity 11

TDI: tolerable daily intake of PCB + PCDD/F DL-PCB + PCDD/F WHO, 1998: EA/MHW (JAP), 1999 SCF (EU), 2001 JECFA (FAO/WHO), 2001 COT (UK), 2010 1 4 pg TEQ/kg b.w./day 4 pg TEQ/kg b.w./ day 14 pg TEQ/kg b.w./week 70 pg TEQ/kg b.w./month 2 pg TEQ/kg b.w./day NDL-PCB 1 pg = 0,000 000 000 001 gram UBA / ex BGA,1983 DFG / OECD TDE, US-FDA 1-3 µg/kg b.w./day 1 µg/kg b.w./day 3 µg/kg b.w./day 1 µg = 0,000 001 gram 12

Derived from dioxin-like toxicity: EU food limit values in fish EU-Regulation 1259-2011 of the Commission (excerpt) 13

Toxicity of PCB in general (1) - the link towards 209PCB Minimization of human intake: Under US/California Drinking Water and Toxic Enforcement Act of 1986 ( Proposition 65 ) In 2006 definition of a general daily dose, regarded as being safe ( safe harbor level ) of 0.09 µg/day total-pcb Special regard for fish oils and derivates for human consumption 14

Toxicity of PCB in general (2) industry: voluntary limit values Industrial (GOED) commitment: limit values for fish oil derivatives EPA, DHA (Eicosapentaenic acid, Docosahexaenic acid = Omega-3-fatty acids) 15

Toxicity of PCB in general (3) 2013: International Association of Research for Cancer (IARC) Béatrice Lauby-Secretan, Dana Loomis, Yann Grosse, Fatiha El Ghissassi, Véronique Bouvard, Lamia Benbrahim-Tallaa, Neela Guha, Robert Baan, Heidi Mattock, Kurt Straif, on behalf of the International Agency for Research on Cancer Monograph Working Group IARC, Lyon, France Carcinogenicity of polychlorinated biphenyls and polybrominated biphenyls The Lancet Oncology Volume 14, Issue 4 2013 287-288 IARC Monograph Volume 107: Polychlorinated Biphenyls and Polybrominated Biphenyls (in press) Official recognition of PCBs (in general) as being human carcinogens (group 1: known carcinogens) 16

PCB analysis: History and Importance 17

PCB-Analysis over the times 1960s: LRGC on packed column); resolution of only about 10 signals for technical pattern. Quantification via technical mixtures as reference 1980s: HRGC (since 1980s capillary columns); detection ECD and LRMS; First assignation of majority of single compounds, pattern analysis Quantification methods for major compounds ( indicator PCB ) 1990s: HRMS; refined quantification; dl-pcb ; isotope-labelled standards; standards for virtually all congeners 18

PCB - analytical methods Discussion of PCB in general implies analysis of a total PCB value Demand for more sophisticated analytical methods for all PCB congeners. Still, present methods are often the old, inaccurate ones or rely upon approximations, e.g. quantification against technical mixtures or analysis of only few marker compounds. Method USEPA 1668 gave a first approach towards a total PCB method The method of choice should be able to cope with all possible analytical demands and answer the client s questions regarding the whole bandwidth of PCBs in-house method deduced from analytical EU reference method for dl/ndl-pcb with elements from US-EPA 1668C 19

Analytical method general flow scheme Sample preparation (e.g. homogenisation) Addition of 13 C 12 -PCB-standards Extraction (e.g. Soxhlet, liquid/liquid, ASE, special) Clean-up 1 (adsorption/ matrix separation) (Silica etc.) Clean-up 2 (adsorption/ fractionation) (aluminiumoxid) PCBs Addition of 13 C 12 recovery standards HRGC-separation HRMS measurement & evaluation 20

Method 209PCB Sample intake: Fish / fish oil: 3 g fat Human blood: 15 g Pigments: 0.05-0.1 g multistep cleanup (adsorption chromatography) HRGC separation: SGE HT8PCB 60m * 0.25 mm i.d. * 0.25 µm df separation of ca. 180 signals wherefrom 140 PCB congeners separated individually and 70 reported as co-elutions (doublets / multiplets) 21

HRMS = High Resolution Mass Spectrometry 22

Method 209PCB /2 HRMS / R 10.000; mass calibration via FC5311 or PFK Isotope dilution quantification: 35 individual 13 C 12 -quantification standards (Mono- through DecaCB) 7 13 C 12 -injection standards (Di- through NonaCB); calibration/identification: all 209 native standards 10 12-point initial calibration; multiple 1-point-calibration daily 23

Method 209PCB /3 QA/QC Performance-driven (responsibility of a lab to see to proper performance and validation) LOQ-determination by S/N ratio and scattering of lab blank data over the whole procedure (avg + 5 stdev.) Instrument LOQ for fish oil at around 1-2pg/g per signal = compound (Mono-TriCB 5pg/g); Total PCB at 400-800 pg total PCB/g (= 0.4-0.8 µg/kg). Method LOQ for fish oil at around typically 5-10 pg/g per signal (compound); Total PCB at 2000-6000 pg total PCB/g (= 2-6 µg/kg). 24

pg TEQ /g 4 3,5 3 2,5 2 1,5 1 0,5 0 2005 2006 2007 2008 2009 2010 Results (1): Total PCB 25

Total PCB: findings Eurofins GfA Lab Service 2014 Informative: Limit value for EPA/DHA (GOED) 26

pg TEQ /g 4 3,5 3 2,5 2 1,5 1 0,5 0 2005 2006 2007 2008 2009 2010 Results (2): PCB congener groups (chlorination degrees) 27

Results - Distribution comparison 28

Results - Congener group distribution raw refined Aroclor 1242 Aroclor 1268 29

Discussion General difficulty due to lack of comparative data (mainly limited to the classic PCB congeners being analysed Difficult for a routine lab due to lack of sample characterisation and missing fat content of original fish in case of oils Comparison on base of single congeners shows a tendency towards lower results possibly due to different origin of samples (open sea catch vs. targeted studies) but: Refined / modified products generally lower than raw fish oils Concentrations and distributions depend on trophic levels Patterns mainly reflect accumulation of higher chlorinated PCBs 30

Other results: colourant samples 31

Results (3): Single congener fingerprints 32

Results / PCB congener distributions 33

Expert knowledge needed: a demanding method!? 34

209 PCB in the laboratory: complex mixtures Complexity mixtures of different kinds / chlorination degrees may cause different analytical performance for single compounds: Overlap of single compounds (co-elution) or with interferences method performance! Use of highly selective instrumentation, e.g. HRMS Ubiquitousity ubiquitous PCB distribution! monitor analytical background Result range! ULTRA trace levels (pg) up to TECHNICAL levels (%) Reduced in food samples (but: accumulation via food chain fish!) QA/QC! 35

Real profile quality assured by use of whole standard set sample 46 69/ 52 73 43/ 49 65/ 75 47/ 48 62 44 59 42 64/ 71 41 68 72 Calibration standard 36

Background effects increased background from PFK/FC5311 Here: DiCB; also Mono-and TriCB affected 37

Only PCBs? interferences Int Hx Hp Hx Hp Hp Hx Int Int Hp Int Hp Sorry, no native #126 employ Carbon cleanup! 38

Summary and conclusions 39

Summary (1) Importance of PCB High total mass of non-disposed PCB General recognition as human-carcinogens by (IARC) Minimization of human intake? legal decisions based on total PCB (US/California Proposition 65 ): safe harbor level of 0,09 µg/day total PCB voluntary limit value for certain products (GOED) Discussion of the whole group of 209 PCB congeners ( total PCB ) 40

Summary (2) Results for Total PCB ( 209PCB ) Analyses have been performed mainly on fish oils / dietary supplements but also on human samples and certain chemical products From the viewpoint of our laboratory, results for fish matrices show the expected differences between raw oils and refined/ modified products. Findings are below the set limit value but still within an important range Levels and distribution of PCB depend on trophic level of the examined species (higher concentrations for higher trophic levels) Still few comparative data available 41

Summary (3) Analytical method Analysis of total PCB and homologue groups: good method performance for limit value control as well as survey of background levels. About 170-190 separations of PCB compounds offer detailed insight into behaviour and distribution of PCB Analysis demanding but only for unexperienced laboratories (volatility of lower PCB; possible co-elutions and interferences; QA/QC; isotope dilution quantification recommended 42

Take Home Analysis of 180 PCB signals instead of 18 dl/ndl-pcb gives you an unique opportunity for having 10 times the trouble! and fun! So, Is it difficult after all? No, but remember the first commandment of ultra trace analysis - THOU SHALT GAIN EXPERIENCE -

Thank you for your attention! Cathrin me Judith The specialtylab / R&D team T: +49 40 49294 5801 FrankNeugebauer@eurofins.de