Human milk sampling

Fundação Oswaldo Cruz
Escola Nacional de Saúde Pública
Centro de Estudos da Saúde do Trabalhador e Ecologia Humana
Global Monitoring Plan
Brazilian Contribution
POPs in Human Milk
Thomas M Krauss
Ana Maria C B Braga
Analytical Capacity
 Sample Preparation: Freeze dryer, Centrifuge
 Extraction: Classic Soxhlet Extractor, Liquid-Liquid
 Cleanup: Open Columns (Biobeads, Silica, Florisil, etc.)
 Instrumental: HRGC/LRMS and HRGC/µ-ECD
 Matrixes:
 Soil (PCDD/F, Indicator PCBs, Pesticides, PAHs)
 Sediment (PCDD/F, Indicator PCBs, Pesticides, PAHs)
 Air Particles (PCDD/F, Indicator PCBs, PAHs)
 Human Milk (Indicator PCBs, Pesticides)
 Human Blood (Indicator PCBs, Pesticides)
 Personal: At the moment only one Chemist for POPs
GMP Phase 1
Second Study 2011 - 2014
Fifth Study of Persistent
Organic Pollutants in Human
Milk coordinated by WHO in
collaboration with UNEP
First study was conducted in 2002 attending the call for participation in the
Third Round of Exposure Studies – PCDD/Fs and PCBs in Human Milk
coordinated by WHO/European Center for Environment and Health
GMP Phase 1
 Contact by the Department for Climate Change and
Environmental Quality of the Ministry of Environment
(MMA), focal point of the Stockholm Convention in
Brazil.
 Project elaboration for monitoring of POPs in Human
Milk in order to fulfill the obligations of the convention
and introduce Brazil in the GMP.
 Approval of
the project e financial support by the
ministry – Collaboration between FIOCRUZ and MMA.
 National Coordination: Ana Maria C. B. Braga e Thomas
M. Krauss.
GMP Phase 1
 Elaboration of the national protocol for human milk sampling
based on the WHO guidelines (2007) and the protocol of the first
study.
 National Protocol used as Base for Standard Operating
Procedure for human milk sampling in the Grulac region.
http://www.ccbasilea-crestocolmo.org.uy/wpcontent/uploads/2010/11/SOP-Regional-de-Muestreo-deCOP-en-Leche-materna.pdf
http://www.ccbasilea-crestocolmo.org.uy/wpcontent/uploads/2010/11/ing1.final_.SOP-POP-RegionalSampling-Breast-Milk-1.pdf
Sampling Locations
First Brazilian Study – Third Round of Exposure Studies
Sampling Locations
Second Study (2011 – 2013) – GMP1
Grande Região 1
Grande Região 2
Grande Região 3
Rio Branco/Acre (3)
Maceió/Alagoas (9)
Brasília/ Distrito Federal
Macapá/Amapá
Salvador/Bahia (10)
Goiás/Goiânia (11)
Manaus/Amazônia
Fortaleza/Ceará
Belo Horizonte/Mina Gerais (12)
Belém/Pará (5)
São Luis/Maranhão (6)
Vitória/Espírito Santo
Porto Velho/Rondônia (2)
João Pessoa/Paraíba (7)
Rio de Janeiro/ Rio de Janeiro (13)
Boa Vista/Roraima (4)
Recife/Pernambuco (8)
São Paulo/São Paulo (14)
Palmas/Tocantins
Teresina/Piauí
Curitiba/Paraná
Cuiabá/Mato Grosso (1)
Natal/Rio Grande do Norte
Porto Alegre/Rio Grande do Sul
Campo Grande/Mato Grosso do Sul
Aracaju/Sergipe
Florianópolis/Santa Catarina (15)
Sampling Strategy - GMP Phase 1
 Volunteer collaboration of human milk banks which are part of the
Brazilian Network of Human Milk Banks
 Seeking of donors (eligibility criteria)
 Application of the questionnaire
 Human milk sampling
 Human milk banks have the following vantages:



Sampling is done by professional that are accustomed with human
milk sampling handling of the samples;
Milk banks have the infrastructure for adequate storage of the
samples;
Professional are aware about the importance of breastfeeding.
 All participating banks were visited and the staff trained for the special
purpose of the study. Delivery of all material (prepared sample bottles,
eligibility guide, consent form, questionnaire and a manual with
detailed instructions.
Type, number and volume of samples
 150 individual samples collected – 100 mL
 15 local pool samples - 350 mL (+ 150 mL reserve)
 analysis of dioxins, PCBs, POPs pesticides and
PBDEs at the reference laboratory
 3 regional pool samples - 750 mL  analysis of all
POPs at the reference laboratory
 150 individual samples - 35 mL  analysis of DDT
and metabolites, and indicator PCBs at the
laboratory of FIOCRUZ.
Sample Shipment
Substâncias analisadas
Compound/Compound group
Regional pool (n = 3)
Local pool (n = 15)
Individual sample (n = 160)
Antigos POPs
PCDDs
SIM
SIM
PCDFs
SIM
SIM
dl-PCBs
SIM
SIM
PCBs indicadores
SIM
SIM
SIM
DDT
SIM
SIM
SIM
Clordano
SIM
SIM
Heptacloro
SIM
SIM
Aldrin
SIM
SIM
Dieldrin
SIM
SIM
Endrin
SIM
SIM
Toxafeno
SIM
SIM
Hexaclorobenzeno (HCB)
SIM
SIM
Mirex
SIM
SIM
Hexaclorociclohexano (HCH)
SIM
SIM
Clordecona
SIM
Hexabromobifenila
SIM
SIM
Pentaclorobenzeno
SIM
SIM
Endosulfam
SIM
SIM
Ácido perfluorooctanosulfônico (PFOS)
SIM
SIM
Polibromodifenil éteres (PBDEs)
SIM
SIM
Novos POPs
Candidatos POPs
Parafinas cloradas de cadeia curta (SCCPs)
SIM
Hexabromociclododecano (HBCD)
SIM
Outras substâncias não POPs
Polibromodibenzo-p-dioxinas (PBDDs)
SIM
Polibromodibenzofuranos (PBDFs)
SIM
Dibenzo-p-dioxinas polihalogenadas (PXDDs)
SIM
Dibenzofuranos polihalogenados (PXDFs)d
SIM
Substâncias perfluoradas (PFCs)
SIM
SIM
Results – PCDDs/PCDFs e dl-PCBs
PCDD/Fs and dl-PCBs Medians - First study 2002
35
pg EQT-OMS1998/g de gordura
30
PCDDs/PCDFs
dl-PCBs
25
20
15
10
5
0
País
Results– Indicator PCBs
Indicador PCBs Medians - First study 2002
550
500
450
ng/g de gordura
400
PCBs indicadores
350
300
250
200
150
100
50
0
País
Results – PCDD/Fs
Average levels of PCDD/Fs found in human milk of de 23 countries in ther period
of de 2008/2009 compared to average level found in Brazil in 2012
Average Brasil 2012:
2,38 pg EQT-OMS2005/g de gordura
2,73 pg EQT-OMS1998/g de gordura
pg EQT-OMS1998/g de gordura
25
20
PCDDs/PCDFs
15
10
5
0
País
Results – dl-PCBs
Average levels of dl-PCBs found in human milk of de 23 countries in ther period of
de 2008/2009 compared to average level found in Brazil in 2012
14
Average Brasil 2012:
0,82 pg EQT-OMS2005/g de gordura
1,14 pg EQT-OMS1998/g de gordura
pg EQT-OMS1998/g de gordura
12
10
dl-PCBs
8
6
4
2
0
País
Results – IndicatorPCBs
Average levels of indicator PCBs found in human milk of de 23 countries in ther
period of de 2008/2009 compared to average level found in Brazil in 2012
90
80
ng/g de gordura
70
60
PCBs indicadores
50
40
30
20
10
0
País
Average = 5,03 ng/g de gordura
GMP 1 Benefits and Problems
 Stimulating the execution of national projects
 Fincancial support – unfortunately low
 Support with consumables, eg HT-8 for PCBs analysis
 Proficiency test
 Complicated bureaucratic procedures for establishing
formal collaborations – international and national level.
 Long sampling times – other sampling strategy?
 Restriction of donors maximum age
Challenges and expectations for the GMP-2 project
 Increase of sample number – sampling in all state capitals of
Brazil including the Federal District – at least 270 individual
samples – maintain three regional pools with 90 individual
samples.
 Involve stakeholders of the Ministry of Health on state or
municipality level – commitment of a local staff for seeking
donors – only sampling and storage at human milk banks.
 Increase laboratorial capacity
 Equipment for analysis of dioxins and other POPs of
complex analysis  HRGC/HRMS and HRGC/MS/MS.
 Contract technicians
Acknowledgement
The study coordinators are grateful to all the coordinators of the participating banks of
the Brazilian Network of Human Milk Banks and their staffs for their voluntary work
and great effort to identify donors and collect human milk samples. Without the support
of these banks it would not have been possible to collect samples all over the national
territory with such a large dimension.
In particular, we thank all the donors for the availability to answer the questionnaire and
the kindness to grant the collection of their milk for this research.
Contacts
Ana Maria C. B. Braga - [email protected]
Thomas M. Krauss
- [email protected]