”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
Clx
4‘
5
6
6‘
5‘
Cly
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
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209 PCB – understanding all PCBs
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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
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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
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209 PCB – understanding all PCBs
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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)
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Global contamination paths
of PCDD/F and dl-PCBs
Bio-Akkumulation
Deposition
PCDD/F
PCB
PCDD/F
PCBEintrag
Emission
Bio-Akkumulation
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209 PCB – understanding all PCBs
Bio-Akkumulation
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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 2,3,7,8Tetrachlordibenzo-p-dioxin
• Long half-lives in human body: PCB 77 0,1a; PCB 126 2,7a; PCB 169 13a
(Liem und Theelen, 1997)
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PCBs: relevance of single congeners
PCBs are a group of polychlorinated aromatic hydrocarbons
3
2
2‘
4
Clx
Cl
ClCl
3‘
Cl
4‘
5
6
6‘
5‘
0,3nm
Cly
Cl
Cl
Cl
Cl
O
x,y = 0...5; x+y>0
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)
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Dioxin-like toxicity: TEF-Concept
(Equivalent-factors acc. to WHO)
highest
toxicity
lowest
toxicity
(TEF human / mammals)
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TDI: tolerable daily intake
of PCB + PCDD/F
DL-PCB + PCDD/F
WHO, 1998:
1 – 4 pg TEQ/kg b.w./day
EA/MHW (JAP), 1999
4 pg TEQ/kg b.w./ day
SCF (EU), 2001
14 pg TEQ/kg b.w./week
JECFA (FAO/WHO), 2001
70 pg TEQ/kg b.w./month
COT (UK), 2010
2 pg TEQ/kg b.w./day
1 pg = 0,000 000 000 001 gram
NDL-PCB
UBA / ex BGA ,1983
1-3 µg/kg b.w./day
DFG / OECD
1 µg/kg b.w./day
TDE, US-FDA
3 µg/kg b.w./day
1 µg = 0,000 001 gram
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Derived from dioxin-like toxicity:
EU food limit values in fish
EU-Regulation
1259-2011 of the Commission
(excerpt)
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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
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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)
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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)
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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
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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
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Analytical method – general flow scheme
Sample preparation (e.g. homogenisation)
Addition of 13C12-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 13C12 recovery standards
HRGC-separation
HRMS measurement
& evaluation
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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)
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209 PCB – understanding all PCBs
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HRMS =
High Resolution Mass Spectrometry
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Method “209PCB” /2
HRMS / R ≥ 10.000; mass calibration via FC5311 or PFK
Isotope dilution quantification: 35 individual 13C12-quantification standards
(Mono- through DecaCB)
7 13C12-injection standards (Di- through NonaCB);
calibration/identification: all 209 native standards
10…12-point initial calibration;
multiple 1-point-calibration daily
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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).
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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)
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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)
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Results Distribution comparison
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Results Congener group distribution
raw
refined
Aroclor 1242
Aroclor 1268
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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
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Other results:
colourant samples
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Results (3):
Single congener fingerprints
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Results / PCB
congener distributions
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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!
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Real profile – quality assured
by use of whole standard set
sample
46
69/ 73 43/
52
49
65/ 47/ 62
75 48
44 59
42
64/ 71 41 68
72
Calibration standard
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Background effects
increased background from PFK/FC5311
Here: DiCB; also Mono- and
TriCB affected
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Only PCBs?
interferences
Int
Hx
Int
Hp Hx Hp
Int
Hp Hx
Hp Int
Hp
Sorry, no native #126 – employ Carbon cleanup!
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Summary and conclusions
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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”)
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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
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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
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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 -
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209 PCB – understanding all PCBs
☺
Thank you for your attention!
Cathrin
me
Judith
The specialty lab / R&D team
T: +49 40 49294 5801
[email protected]