non-invasive biomonitoring for frs - university of birmingham...serum present -large volume of...
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Non-invasive biomonitoring for FRs
Dr. Adrian Covaci
Toxicological Center, UA
INFLAME – ATC2 – Thu 15-09-2011, Antwerp
Summary
- Generalities – non-invasive matrices
- Hair physiology (specific issues related to organic compounds) - Hair biomonitoring for persistent organic pollutants (POPs)
- flame retardants - Advantages and shortcomings for hair as non-invasive biomonitoring matrix for organic compounds - Other applications of hair (animals) - Other keratinous matrices
- bird feathers for wildlife biomonitoring - human nails
- Other non-invasive matrices for biomonitoring (urine!!)
Research questions
-Why biomonitoring
- Which matrices are regularly used? (serum and milk)
- Advantages and disadvantages?
Disadvantages
- rather invasive,
- informed consent mandatory
- milk – only women (18-40y)
- serum difficult to samples
from children
Advantages
- many current programs
- database on these matrices
- easy to handle and process
Summary
Which are matrices are “non-invasive”? Hair Urine Nails Skin or skinwipe Earwax Saliva
Information from different matrices
Matrix Exposure Comments
Serum Present -large volume of sample needed
Urine Present - mostly soluble and/or
metabolizable compounds
Milk Past -integrative data
available from sub-populations
Adipose tissue Past -integrative data
Organs (liver, brain) Present -invasive specimen
Hair Past-internal -allows sequential analysis
Present-external
Biological matrices vs detection windows
Min. Hours
Days
Weeks
Months
Years
Blood
Oral Fluid
Urine
Sweat
Hair
Organics in hair
Physiology of hair
- An appendage of the skin that grows out of an organ known as the hair follicle
- Extends from its root or bulb embedded in the follicle, continues into a shaft, and terminates at a tip end.
Types of hairs
• Head hair - Long with moderate shaft diameter and diameter variation - Often with cut or split tips - Can show artificial treatment, solar bleaching, or
mechanical damage - Soft texture, pliable
• Pubic hair - Stiff texture, rigid
• Facial hairs (beard/mustache) • Chest hair • Other hairs (eyebrow)
Hair Root
• Provide the tools to produce hair and continue its growth • 3 Stages of Growth (different looking roots)
- Anagen -initial phase may last up to 6 years, root is flame shaped
- Catagen –transition phase (2-3 weeks), root is elongated - Telogen –phase where hair naturally falls out of the skin
Hair - general information
Hair = Annex of the skin originated in hair follicle
Hair follicle : cells in active proliferation
Hair shaft: keratinized cells with different layers
including cuticle and medulla
Growth ~1 cm/month
85% in growth phase
1% in transition phase
14% in dead phase
composition 88% proteins (S-S bonds between chains) - keratins
1-4 % lipids (FFA, TG, cholesterol)
water, minerals, melanins
Internal exposure - compounds present in the blood and capsulated in the
hair follicle
External exposure - compounds present in the air and solubilized in the
sebaceous glands excretions
Standardization of hair sampling
• Questioned and Reference hair must come from same area of the body
• The collection should ensure the representativeness of the sample.
• All collected hairs must be full-length to check if the color and morphological features vary throughout the length of the hair.
• If possible, use tweezers to pick up hair, store it in paper envelopes.
• Last, but not least, ENOUGH sample must be collected
Models proposed for incorporation in hair of organic compounds
1. Diffusion from blood into growing cells in hair follicle
2. Idem 1 + diffusion from body secretions (sweat, sebum,…) during or after shaft formation
3. Idem 2 and/or external environmental sources after hair shaft formation
Cuticule Cortex Medulla
2- Sweat, Sebum
3- Environment
1- Blood
Hair - cut
- powder - Micro LSE
- SPE
Clean-up
Quantification
General scheme of hair analysis
Organic
extract
Cleaned
extract
GC-MS
LC-MS
Incubation Decontamination + IS
-Removal of
external
contamination
-Denaturation of the
keratinous matrix
-Removal of
matrix
interferences
Methods for BFRs in hair
Decontamination - removal of dust and soil particles - removal of external contamination (e.g. for volatile
compounds from air)
Homogenization - Powdering in ball mills - Cutting Incubation - With acid (HCl, H2SO4, etc) - With base (NaOH) - Enzymatic (protease)
Extraction SLE – with organic solvents (MeOH, hexane, DCM, acetone) SPE (suitable adsorbents) Soxhlet Clean-up Elimination of large molecules (proteins) and lipids
Multi-layer cartridge - silica based adsorbents (acidified, activated) - Florisil, alumina - carbon
- SPE
Elution with suitable organic solvents
Methods for BFRs in hair
Detection GC-LRMS - EI - CI GC-MS/MS GC-HRMS
LC-MS (or LC-MS/MS)
Methods for BFRs in hair
Other possibilities : - HPLC fractionation - Large volume injection in GC
QA/QC
- Indicative values for PCBs, OCPs and PBDEs - Material is discontinued!! - IRMM is currently certifying a new hair material for ..metals only.. .
POPs in hair
- Much less information available about BFRs
• Very few publications deal with establishing relations between the levels of POPs in paired human hair and blood.
- Moderate to strong correlations between the hair and blood levels were reported only for ppDDE, ppDDT, PCB 28 and PCB 74 (Altshul et al., 2004).
- Similar correlations found for PCDD/Fs and coplanar PCBs (Nakao et al., 2002).
POPs in hair
POPs in hair
Polychlorinated and polybrominated compounds
Zhao et al., Environ Int 2008
FRs in hair
Dechlorane Plus -Hair and dust -Resident and e-waste workers
- hair (0.02-58.32 ng/g)
- dust (3-4197 ng/g)
FR in hair samples
Average percentage distribution of PBDEs, DBDPA, BTBPE, HBB and PBBs in Chinese hair samples (Zheng et al., Environ Pollut 2011)
PBDE distribution in - Newborn hair - profile from in utero exposure from the mother) - Child hair – profile from post-natal exposure (breast-feeding, dust and diet)
Aleksa et al., SETAC Europe 2011
PBDEs in hair
Ranges of concentrations in various studies 2005 - China (dismantling women workers in e-waste) – 10 – 490 ng/g dw 2006 - China (dismantling workers in e-waste) – 870 ± 205 ng/g dw 2007 - China (dismantling workers in e-waste) – 5 - 100 ng/g dw 2008 – Spain – general population – 1.5 – 20 ng/g dw
PBDEs in hair
Profile PBDEs in dismantling workers
Advantages of hair testing
• Cost-effective and time-saving (one hair test vs. approximately 18 urine samples for a 3-month profile).
• Larger detection windows (from 3 days to years), depending on the length of the hair shaft; urine/blood (hours to days for most drugs);
• Evaluation of long term history to short term history
• Sample collection is non-invasive, it is easy to be performed under conditions that prevent adulteration
• Simpler to collect, store and transport.
• Excellent pre-employment screening tool.
• Applicability in large monitoring (screening) studies
• Cheap and miniaturised methods
• Small amounts of sample (drugs 20-50 mg, POPs 200 mg-10 g)
CRITICAL ISSUES
Interpretation of results
• limited correlation between the ingested dose and the concentration in hair
• inter-individual differences in dosage and thus serum concentrations
• limited knowledge of excretion and distribution of POPs in hair
• differing compound incorporation rates, influenced by hair pigmentation and physical state of the hair (shampooing, bleaching, dyeing, permanent wave).
Validation • Heterogeneous matrix
• Stability of drugs
• Influence of sample preparation
• No certified reference materials available
Use of NEONATAL HAIR
Cumulative exposure to drugs during the last trimester of intrauterine life
Neonatal hair vs meconium:
• Advantages:
◊ hair has the advantage of being available for as long as 4 to 5 months of postnatal life
• Limitations:
◊ detection window for neonatal hair is smaller than for meconium
◊ hair samples from newborns are often sparse, and collection can be considered „„almost‟‟ invasive
Has been used for drugs, but also pesticide exposure, not yet for BFRs
Hedghehogs (Erinaceus europaeus) - Belgium
- Dead hedgehogs (D’Havé et al., Environ Sci Technol 2005), n = 43 individuals (mostly
road kills)
Purpose: investigating relationships between PBDE concentrations in hair and in various tissues (liver,
muscle, kidney, fat)
Findings
- positive relationships between lconcentrations in hair and internal tissues for sum PBDEs and BDE 47
(0.37 < r < 0.78).
- values of correlation coefficient are compound-specific!
Hair use for biomonitoring of PBDEs in wildlife
Hedghehogs (Erinaceus europaeus) - Belgium
Study 2. Living young hedgehogs (Vermeulen et al., Environ Int 2010), n = 20 individuals
POPs: PCBs, DDTs, HCHs, HCB, PBDEs
Purpose:
- investigating relationships between POP concentrations in hair and blood of young hedgehogs
- Investigate the uptake of POPs through the food chain
Findings
- no relationships between concentrations in hair and blood
Hair use for biomonitoring of POPs in wildlife
Polar bear (Ursus maritimus) – East Greenland
Study 4. Dead polar bears
(Jaspers et al., Sci Total Environ 2010) n = 12 individuals
Sample size: 15 – 130 mg
POPs: PCBs, DDTs, HCHs, HCB, PBDEs
Purpose: investigating relationships between POP concentrations in hair and in various tissues (liver,
muscle, kidney, fat)
Findings
- positive relationships between POP concentrations in hair and organs (r = 0.40 - 0.90) for most
POPs
- higher corellation coefficients between hair and adipose tissue or liver, compared to blood
Hair use for animal biomonitoring
Other keratinous matrices
FEATHERS
Buzzard (Buteo buteo) – Belgium
Study 1. Dead buzzards (Jaspers et al., Biol Lett 2007) n = 43 individuals
Sample size: 400 – 700 mg
POPs: PCBs, OCPs, PBDEs,
Purpose: investigating relationships between POP
concentrations in feathers and in various tissues
(liver, muscle, kidney, fat)
Findings
Other keratinous matrices
FEATHERS
Goshawk, Sea eagle and golden eagle – Northern Norway
Study 2. Living raptors (Eulaers et al., Environ Int 2011) n = 40 individuals/year
Sample size: 400 – 700 mg, NESTLINGS
POPs: PCBs, OCPs, PBDEs,
Purpose: investigating relationships between POP concentrations in feathers and blood of nestlings
Findings
-Body feathers have higher concentrations than wing or tail feathers !!
- good correlations (r > 0.7) between conc in feathers and blood for most POPs
Other keratinous matrices
Nails (finger, toe) for humans
Dioxin 2010, St. Antonio, US, 12-17Sept 2010
- 0.1-0.4 g available per clipping turn
- need to homogenize (grind?) thoroughly the nail samples
- no information available regarding the distribution of organic
pollutants between blood (or other tissues) and nails
Urine
Matrix of choice for:
- polar analytes (are there such between FRs?)
- metabolites (mostly phase I, but also phase II)
Examples:
- dialkyl or diaryl phosphates (DAPs) as metabolites of trialkyl of
triaryl phosphates (TAPs)
- HO-PBDEs (?) and bromophenols as metabolites of PBDEs
Urine
Examples: TAP – DAP – MAP
Urine
Metabolism of PBDEs
-Hydroxilation
-Debromination
-Formation of bromophenols
-In vivo and in vitro studies!
-Metabolism of HBCDs
-Hydroxylation
-Debromination
Urine
Right matrix – to reflect exposure
Right analyte – to reflect exposure
- Parent? Metabolite? Phase I? Phase II?
Right analytical technique – to be able to measure the target
analyte
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