Monitoring
of Persistent Organic Pollutants
in human milk and blood
Anton Kočan, Jana Klánová

Matrices selected for the GMP
§The COP has decided that the air monitoring and human exposure through breast milk or maternal
blood will be used as core media for the first evaluation planned in 2009.
§For future evaluations, the COP has also decided to endeavour to supplement the core data with
data from other media such as biota, water, soil, and sediments.
A. Kočan, Slovak Medical University

What is Human Biomonitoring (HBM) ? (1)
§HBM is the assessment of human exposure to chemicals present in air, water, soil, dust, food or
other environmental media via measurement of the chemicals or chemical metabolites present in human
specimens, such as blood, urine, hair, saliva, feces, tissues. The result of such measurement is
usually called „body burden“.
§HBM is a tool to help better understand human exposure to environmental chemicals – both natural
and man-made.
§If gathered from a representative sample of a population – for instance, children or adults in a
particular area – HBM can be used to document whether that subgroup as a whole has been exposed to
some chemicals and to what extent.
A. Kočan, Slovak Medical University

What is Human Biomonitoring (HBM) ? (2)
§HBM is a scientific technique for assessing human exposures to environmental agents and their
effects, based on sampling and analysis of an individual´s tissues and fluids. While blood, urine,
breast milk and expelled air are most commonly measured, hair, nails, fat, bone and other tissues
may also be sampled.
§This technique takes advantage of the knowledge that environmental agents that have entered the
human body leave markers reflecting this exposure. The marker may be the agent itself or a
breakdown product, but it may also be some change in the body resulting from the interaction of the
agent or its breakdown product(s) with the individual, such as alterations in the levels of certain
enzymes or other proteins which may lead to modifications of normal body processes.
A. Kočan, Slovak Medical University

HBM can be used to :
§Reinforce regulatory actions by providing actual data about which chemicals get into people and at
what levels.
§Improve exposure assessment.
§Establish baselines or reference ranges.
A. Kočan, Slovak Medical University
§Facilitate people's right to know what chemicals are in their bodies.
§Establish priorities for tackling environmental health-related problems.

Some variables affecting HBM (2)
§The precision of chemical analysis is generally believed to constitute only a minor part of the
total variance in monitoring time-series of environmental data where sample variation is expected
to be large, much larger compared to laboratory precision.
§That is true if the same accredited laboratory is used through the whole series. However, if, from
year to year, different laboratories carry out the analysis, it could seriously decrease or disable
the possibility to evaluate time-series of, for example, POPs.
A. Kočan, Slovak Medical University
§The same is true if the same laboratory changes its methodology and, for example, co-elutions are
resolved leading to a decrease in estimated concentrations unless measures are taken to compensate
for them.
§If detection limits are improved, i.e. analytes are now found where they were not detected before,
that may lead to similar problems depending on how results below the limit of quantification (LOQ)
are treated.

Some variables affecting HBM (1)
§Fat content and composition in human milk changes dramatically during the first weeks after birth,
which leads to variation also in analysed POPs. In order to reduce random variation, sampling
should preferably be carried out during a well defined period, e.g. 3 weeks after birth. Also the
fat content varies considerably depending on whether sampling is carried out in the beginning or at
the end of the feeding session.
§The use of narrow sampling unit definition implies that a smaller part of the studied population
is represented. Often, this leads to unfounded assumptions of similar trends, e.g., for both sexes
or for various age classes. To improve representativity, if economy permits, stratified sampling
should be applied rather than allowing for a wider definition of the sampling unit.
A. Kočan, Slovak Medical University

Ethical issues relevant to HBM
§Ethical issues are relevant in almost every facet of human biomarker research from the design of
the studies, the identification and recruitment of subjects, to the handling and use of the data,
and interpretation and communication of the results.
§The right to monitor chemical pollutants in blood and breast milk is a crucial aspect of community
right to know but also brings with it responsibilities to care and support those who are tested. .
§Researchers and regulators have to be aware of the potential for biomarker information to affect
the lives of subjects and their families. There must be sufficient protection of personally
identifiable data and regulation of its use, while ensuring individual subjects right to know their
results.
A. Kočan, Slovak Medical University

Number of samples needed
§More samples provide more precise and reliable estimates of mean concentrations and variance.
However, the contributions from additional samples depend to a very high degree on the sampling
strategy.
A. Kočan, Slovak Medical University
§Using pooled samples of several specimens will decrease the number of chemical analyses required
to estimate a reliable mean concentrations compared to one or a few individual samples, since a
larger proportion of the total population is represented.
Disadvantages with pooled samples are that extreme values from single specimens may influence the
concentration of the pool without being revealed.

§All sampling should follow established methodological guidelines, which should be agreed upon
before the start of any programme activity in a region.
§If possible, samples in all programmes should be numbered in the same way.
A. Kočan, Slovak Medical University
General considerations pertaining to GMP sampling (1)
§The sampling window for the initial baseline will be 2003 ± 5 years.
§Sample frequency and timing should, as much as possible, be harmonized between matrices.
As a rule, samples should be taken at least annually and during the same period every year.
For some matrices where seasonal influences would be less important (e.g. human breast milk), the
sampling frequency and duration might be different.
For the statistical analysis of the levels it would be preferable to take many samples frequently
from one location rather than to take a few samples from many different locations.

A. Kočan, Slovak Medical University
General considerations pertaining to GMP sampling (2)
§Sampling should always include field or trip blanks and, to the extent possible, duplicate samples
for the purpose of sample sharing and the analysis of variance.
§Sample banking should be considered for all samples. Sample banking is an expensive and resource
intensive activity that needs to be sustainable in a long time perspective. However, if properly
managed it may yield important insights into exposures over time (e.g for new POPs) and may also be
used for retrospective studies.
P1240002 P1240004
A sample bank at the Slovak Medical University in Bratislava

Human milk and maternal blood as matrices for the GMP (1)
§Human milk and maternal blood are both good sample media for assessing POPs exposure in humans.
Furthermore, both these media can be used to demonstrate possible temporal trends and regional
variations in levels.
§Human milk sampling is non-invasive and milk can generally be obtained from lactating mothers in
reasonable quantities. In certain populations it may, however, be difficult to obtain human milk
samples in the required time period, i.e., 2 to 4 weeks after delivery.
§Blood sampling is invasive, but sampling of mothers prior to giving birth may readily be achieved.
However, blood sampling may not be acceptable in certain cultures.
A. Kočan, Slovak Medical University
§Depending on local considerations, biological samples of human origin, including blood and milk,
should be considered a potential biohazard. Necessary precaution procedures should be applied to
both sampling and handling of all samples, not only in situations where one may expect a problem,
e.g. HIV-positive serology and hepatitis.

Human milk and maternal blood as matrices for the GMP (2)
§The limit of detection for POPs will in general be lower in milk than in blood.
The reason for this is partly the difference in lipids between the media and the fact that larger
volumes of milk as compared to blood can normally be obtained.
When the limit of detection is approached the analytical precision will decrease.
§An important consideration in the choice of human milk and maternal blood as biological indicators
is that we will only obtain information from a specific part of the population both with regard to
gender and age.
As for future trend studies, a careful evaluation should be done to explore alternative
representative groups in a population, e.g. men (specified age groups), youth groups of both
gender, school children or infants.
A. Kočan, Slovak Medical University

Human milk and maternal blood as matrices for the GMP (3)
§A population study must be based on sampling and analyses of individual samples; human milk or
maternal blood.
Pooled samples might be considered for certain contaminants, such as the dioxins which are
expensive to analyze and need larger sample volumes.
§In order to reduce sample variance and facilitate comparability a stratified sample design should
be adopted.
This should be based on demographic information collected in specific questionnaires, i.e. age,
residence, occupational history, smoking habits, current and previous diet, etc.
A. Kočan, Slovak Medical University
§Selection of study groups should be based on known exposure patterns, global or local.
The groups with known high exposure levels are more sensitive to changes in the environment and
will provide better indications in trend analyses. Even in countries with very limited background
information one might be able to select population groups of interest, such as rural versus urban;
fish eating populations versus rural agricultural populations with high exposures to pesticides;
populations living in areas with re-introduction of DDT for malaria prophylaxis etc.

Human milk and maternal blood as matrices for the GMP (4)
§Sample size will depend on the circumstances, and to estimate the number of samples needed a
number of factors have to be considered to achieve representative samples. For either human milk or
blood, 50 individual samples are to be collected. However, new technologies and new, certified
laboratories will provide the opportunity to begin epidemiological studies with individual results
on a larger scale.
§The choice of milk or blood depends very much on the practical implementation regionally or
locally. Two examples:
A. Kočan, Slovak Medical University
üIn the Arctic many indigenous women deliver their babies and go home to the tundra before they
have started their milk production. To collect colostrum provides a very different medium than the
fully developed breast milk 2-3 weeks after delivery. It is not possible to trace the women at the
right time for breast milk collection. A blood sample will solve that problem.
üIn certain areas of Africa sampling of maternal blood might be problematic. In those cases breast
milk is the best matrix.
§Trained personnel is crucial at the sampling and analytical stages. Standardized protocols,
equipment and education of field personnel as well as laboratory personnel must be implemented.

Human milk and maternal blood sampling
§Human milk should be sampled according to a WHO protocol.
§At least 50 individual milk (blood) samples should be collected. But countries with populations
over 50 million should include at least one additional participant per one million population over
50 million. Countries with populations well over 50 million (or with sufficient resources) are
encouraged to prepare a second pooled sample (or more) if feasible.
A. Kočan, Slovak Medical University
üMother should be primiparae
üMothers who may have unusually high exposure to POPs (living in the vicinity of incinerators, pulp
or metal industiries or where organochlorine chemicals have been produced) should not be included
in the survey to avoid skewing the results.
§Maternal blood should be sampled according to an AMAP protocol.
§Selection criteria for mothers:
üMother should be under 30 years of age
üBoth mother and child should be apparently healthy, including normal pregnancy
üMother should be breastfeeding one child only (i.e., no twins)
üMother should have resided in the area for at least the previous 10 years
[USEMAP] [USEMAP]

How to identify possible donors (1)
A. Kočan, Slovak Medical University
üPossible donors can be contacted before giving birth In countries with adequate pre-natal
coverage.
§Selection before giving birth:
üAll potential donors should be informed about the benefits of breastfeeding and be encouraged to
breastfeed even if they do not intend to or are not selected to participate in the survey.
üOnce a participant indicates a willingness to take part in the survey, she should be invited to
complete certain section of the questionnaire.
The questionnaire can be completed through a personal interview at the pre-natal clinic or
completed by the potential donor at home and returned to the clinic, either in person or by mail.
üDepending on the homogeneity of the population, up to 250 completed questionnaires should be
collected. Depending on the country, more than 50 potential and 10 reserve donors should be
selected to take into account possible withdrawals. Participants should be notified of their
selection and where and when the sample will be collected.

How to identify possible donors (2)
A. Kočan, Slovak Medical University
üSamples are collected of mothers at postnatal clinics and other venues, e.g., well-baby clinics
without pre-selection.
§Selection after giving birth:
üMothers can be contacted directly and interviewed to complete all section of the questionnaire.
üSamples can then be collected immediately or at another appropriate time.
üWhile this method can reduce the time of the survey by up to 3 months, it does not allow for
further stratification of the cohort to reduce variability.
üHowever, after the cohort selection criteria have been established from the first sample
collection, this method offer advantages for the second and subsequent sample collections.

Collection of samples
A. Kočan, Slovak Medical University
§Sampling can be carried out between 3 to 8 weeks after delivery.
§At least 50 ml of milk in total should be collected by hand expression after a feeding or while
infant is nursing on the other breast, to take advantage of the let-down reflex of the mother.
A human milk pump to facilitate expression can be applied.
If necessary, the mother may collect the sample at home, in which case manual expression is
preferred. If so, she should be given detailed instructions for sampling, storing and transporting
of milk samples.
Mothers should also be given a clean glass jar with a protected screw cap to collect and store the
milk sample.
§The sample should be collected directly to the collecting jar and, if collected at home, stored in
the collecting jar in the home freezer until it can be delivered.
Otherwise milk samples may be stored in the refrigerator at about 4 °C for a maximum of 72 hours.
If refrigeration is not available, a tablet of K2Cr2O7 may be added to chemically sterilize the
milk. If the milk is to be collected at home, the tablet may be placed in the collection jar before
it is given to the donor.
CAUTION: The mother must be told to keep the jar with potassium dichromate away from other children
in the household as this is a toxic chemical.
[USEMAP]

Questionnaire for mothers donating breast milk (1)



Questionnaire for mothers donating breast milk (2)



• SPE (10 g C-18 column), 10 - 30 mL serum
PCDD/PCDF/PCB analysis (1)


HPIM2301 HPIM2300
C-18 column
Blood serum

• SPE (10 g C-18 column), 10 - 30 mL serum
•Clean-up using semi-automated equipment (H2SO4/silica, alumina, carbon chromatography) :
PCDD/PCDF/PCB analysis (1)

HPIM2302
Semi-automated sample cleanup system


HPIM2303
Hexane eluate
from an SPE column
Silica/H2SO4
layer
Silica/KOH
layer
Alumina column
Carbon column

• SPE (10 g C-18 column), 10 - 30 mL serum
•Clean-up using semi-automated equipment (H2SO4/silica, alumina, carbon chromatography) :
•Fraction 1:
•Fraction 2:
•WHO mono-ortho-PCBs (105, 114, 118, 123, 156, 157, 167, 189)
•other ortho PCBs (18, 28, 44, 49, 52, 66, 74, 87, 99, 101, 110, 128, 138, 146, 149, 151, 153, 170,
172, 177, 178, 180, 183, 187, 194, 195, 196/203, 201, 206, 209)
•Seven 2378-PCDDs
•Ten 2378-PCDFs
•Four coplanar PCBs (77, 81, 126, 169)
PCDD/PCDF/PCB analysis (1)

PCDD/PCDF/PCB analysis (2)
•1 analysis batch is composed of:
•14 serum samples
•1 in-house RM (spiked porcine serum)
•1 blank (no serum)
•occasionally CRM (serum)
•HRGC (DB5-ms) separation
•HRMS (10000 res) quantification
•Isotope dilution method based on EPA 1613 and 1668

•
0
2
4
6
8
10
12
14
16
18
20
PCDDs/PCDFs
PCBs
European comparison of PCDD/F and PCB levels in Human milk (WHO Exposure Study 2001/2002)
Source: Van Leuwen, R. Malish 2002 "WHO exposure study on the levels of PCBs, PCDDs and PCDFs in
Human Milk" 3rd round
A. Kočan, Slovak Medical University

0
5
10
15
20
25
30
35
40
Time trends in PCDD/F levels in human milk
(WHO Exposure Studies)
Source: WHO
A. Kočan, Slovak Medical University
1988
1993
2002
2007
Source: Van Leuwen, R. Malish 2002 "WHO exposure study on the levels of PCBs, PCDDs and PCDFs in
Human Milk" 3rd round
A. Kočan, Slovak Medical University

Slovakia‘s districts chosen for exposure studies
A. Kočan, Slovak Medical University
Mapa_okresy

Time trends in PCB levels in human blood
(2 districts in Slovakia)
A. Kočan, Slovak Medical University
0
500
1000
1500
2000
2500
3000
3500
4000
4500
PCBs
HCB
pp´-DDE
 MI 1997 (215 blood samples)
 MI 2001 (1011 blood samples)
 SP 1997 (205 blood samples)
 SP/SV 2001 (1038 blood samples)

“Cons” - Why is Public Health Biomonitoring  Controversial?
§May cause unfounded fear. Because it can be measured, doesn’t mean it causes harm.
§Hard to tell how much  is too much. No “meaning” without  reference ranges.
§Provides no information about sources.
A. Kočan, Slovak Medical University
§May modify behavior in negative ways.
§May cause valuable products to be banned.

“Pros” - Biomonitoring helps answer
Important Public Health Questions
§What are we exposed to and how much?
§Do public health policies and regulatory programs reduce exposures over time?
§Are some groups more highly exposed than the general population?
A. Kočan, Slovak Medical University
§What are the relationships between exposure and disease?
Source: P. Flessel, EHL, California DHS

Why to breastfeed
§Nutrition:
A. Kočan, Slovak Medical University
üBreast milk provides, in an easily digested form, all the nutrients an infant needs for the first
six months of life. Exclusive breastfeeding (i.e., no other food or drinks given, not even water)
for the first six months offers maximum protection to infants against pneumonia, diarrhoea and
other common infections of childhood.
üUp to 2 years of age or more, breast milk continues to provide high-quality nutrients and helps
protect against infection.
From 6 to 12 months, breast milk usually provides 60–80% of all energy, protein and other
nutritional requirements – e.g., vitamins and other micronutrients.
From 12 to 23 months, breastfeeding can provide up to 35–40% of these requirements.
§Psychosocial development:
§Family planning / child spacing:
üBreastfeeding delays the return of a woman’s fertility. A woman who does not breastfeed is at
increased risk of becoming pregnant again as early as six weeks after the birth of the child.
üBreastfeeding promotes the emotional relationship, or bonding, between mother and child.

Slovakia‘s Districts Chosen for POPs Exposure Studies:
Nitra
Bratislava
Myjava
Trebišov
Michalovce
Veľký Krtíš
in 1992 - 94
Mapa_okresy
in 1997
Mapa_okresy
in 2001
&
2003-05
A. Kočan, Slovak Medical University

Michalovce2s
A. Kočan, Slovak Medical University


Michalovce District
Stropkov District
PCB Concentrations in Ambient Air
Michalovce (polluted district) vs Stropkov (control district)
110
110
100
280
1700
1500
200
180
73
80
85
64
Senne
Hazin
Michalovce I
Michalovce II
Vola
Strazske
Stropkov I
Stropkov II
Potocky
Brusnica
Lomné
Turany n/O.
PCB Concentration [pg/m
3
]
A. Kočan, Slovak Medical University

PCB Concentrations in Soil
Vicinity of Chemko disposal sites and agricultural fields
Waste Disposal Sites
Agricultural/Forest Soils
A. Kočan, Slovak Medical University

Michalovce District
Stropkov District
PCB Levels in Sediment Samples
Michalovce (polluted district) vs Stropkov (control district)
3 000 000 = 0.3 %
A. Kočan, Slovak Medical University

PCB Levels in Fish
Michalovce (polluted district) vs Stropkov (control district)
Stropkov waters
Michalovce waters
A. Kočan, Slovak Medical University

Average PCB Levels in Eggs and Chicken
Collected in Michalovce and Stropkov Districts
Sum of 28, 52, 101, 118, 138, 153, 156, 170 and 180 congeners
24,5
15,4
18,3
16,2
374,3
128,9
2764,2
Chicken (SP-retail)
Eggs (MI-producer)
Eggs (MI-retail)
Eggs (SP-retail)
Chicken (MI-
retail+home)
Eggs (SP-
homemade)
Eggs (MI-
homemade)
ppb (lipid basis)
A. Kočan, Slovak Medical University

0
20000
40000
60000
80000
100000
15
20
25
30
35
40
45
50
55
60
65
70
75
0,0
20000,0
40000,0
60000,0
80000,0
100000,0
15
20
25
30
35
40
45
50
55
60
65
70
75
Age
Svidnik+Stropkov
Michalovce
ng/g, lipid adjusted
PCB Levels - Adults
A. Kočan, Slovak Medical University

0
20000
40000
60000
80000
100000
15
20
25
30
35
40
45
50
55
60
65
70
75
0,0
20000,0
40000,0
60000,0
80000,0
100000,0
15
20
25
30
35
40
45
50
55
60
65
70
75
Age
Svidnik+Stropkov
Michalovce
ng/g, lipid adjusted
PCB Levels - Adults
Husband (78)
Wife (72)
both ate local fish
A. Kočan, Slovak Medical University

0
20000
40000
60000
80000
100000
15
20
25
30
35
40
45
50
55
60
65
70
75
0,0
20000,0
40000,0
60000,0
80000,0
100000,0
15
20
25
30
35
40
45
50
55
60
65
70
75
Age
Svidnik+Stropkov
Michalovce
ng/g, lipid adjusted
PCB Levels - Adults
Woman (55)
Woman (70)
high portion of food
from home-kept animals
A. Kočan, Slovak Medical University

•
Median PCB-153, HCB, and p,p’-DDE levels in blood serum vs age groups (specimens collected within
the PCBRISK project in 2001
1665
1056
718
348
151
90
994
367
364
295
212
109
4262
2485
1967
1361
763
473
60 – 78
50 – 59
40 – 49
30 – 39
17 – 29
8 – 9
 PCB-153
 HCB
 pp'-DDE
ppb, lipid adjusted
N=434
N=369
N=198
N=708
N=683
N=89

Comparison of POP levels in men and women and time trends
1998
Study
1998
Study
2001
Study
2001
Study
A. Kočan, Slovak Medical University

v 32- and 4.3-fold increase of DDT and DDE respectively in a mother before delivery vs the mother
21 months later
v 180- and 42-fold increase of DDT and DDE respectively in her 16-m child vs the 6-m child
v 35-% decrease of DDT in the child at the age of 21 moths while DDE unchanged
pp'-DDE and pp'-DDT levels in the blood of a mother and her child (#4318)
1020
864
96
4023
4345
4023
67
48
23
4075
2121
2694
0
500
1000
1500
2000
2500
3000
3500
4000
4500
pp´-DDE
pp´-DDT
Mother‘s and child‘s blood was taken
when the child was 21 months old
A. Kočan, Slovak Medical University

ng/g, lipid adjusted
PCB-153 congener levels in blood serum taken from
mothers and their umbilical cords (141 pairs)
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
Mothers
R=0.967
A. Kočan, Slovak Medical University

ng/g, lipid adjusted
PCB-153 congener levels in blood serum taken from mothers
and their 6-month-old children (141 pairs)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
Mothers
R=0.583
A. Kočan, Slovak Medical University

ng/g, lipid adjusted
PCB-153 congener levels in blood serum taken from children
at the age of 6  and 16 months (141pairs)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
16-month-old children
R=0.916
A. Kočan, Slovak Medical University

PCB Levels in Human Milk Samples / WHO
Sum of 28, 52, 101, 138, 153, and 180 congeners
0.25
0.49
0.40
0.33
0.49
0.62
1.02
Slovakia 2006
Kosice_MWI 2001
Svidnik 2001
Nitra 2001
Nitra 1993
Michalovce 2001
Michalovce 1993
pg/g, lipid adjusted
A. Kočan, Slovak Medical University

PCDD/F-TEQ Levels in Human Milk Samples / WHO
2.5
3.1
4.2
3.7
4.2
3.5
3.4
4.0
4.8
5.4
4.9
8.5
6.4
11.7
10.1
12.5
12.7
10.7
19.5
Slovakia 2006
Kosice_MWI 2001
Svidnik 2001
Nitra 2001
Nitra 1993
Michalovce 2001
Michalovce 1993
pg/g, lipid adjusted
PCDDs
PCDFs
dl-PCB
A. Kočan, Slovak Medical University

A. Kočan, Slovak Medical University
Recommended Literature & Links
Pertaining to Human POPs Monitoring
§Guidance on the Global Monitoring Plan for Persistent Organic Pollutants. UNEP. February 2007
(preliminary version).
§Guidance for Analysis of Persistent Organic Pollutants (POPs). UNEP. March 2007.
http://www.chem.unep.ch/pops/laboratory/analytical_guidance_en.pdf
§Guidelines for Developing a National Protocol. WHO. March 2007 (revised).
http://www.who.int/foodsafety/chem/POPprotocol.pdf
§http://www.pops.int
§http://www.chem.unep.ch/pops/default.html
§http://www.cdc.gov/biomonitoring
§http://www.aphl.org/programs/environmental_health/biomonitoring/Pages/default.aspx
§http://www.who.int/foodsafety/chem/pops/en/
§