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