Standard Operating Procedure for POP Sampling in Breast Milk

Standard Operating Procedure for POP
Sampling in Breast Milk
UNEP-GEF Project
Project to Support the Implementation of the Global Monitoring
Plan for Persistent Organic Pollutants (POPs) in the Countries
of Latin America and the Caribbean (LAC)
GFLIPMS 3778
July 2010

Standard Operating Procedure for
POP Sampling in Breast Milk
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Table of Contents
1. INTRODUCTION ............................................................................................ 2
2. DOCUMENTS OF REFERENCE ................................................................... 3
3. OBJECTIVES ................................................................................................. 3
4. SCOPE ........................................................................................................... 3
5. METHODOLOGY ........................................................................................... 4
5.1 TYPE, NUMBER AND VOLUME OF SAMPLES ...................................................... 4
5.2 COLLECTION OF SAMPLES ............................................................................. 4
5.3 TRAINING OF COLLABORATORS ...................................................................... 5
5.4 SELECTION OF DONORS AND IMPLEMENTATION OF QUESTIONNAIRE .................. 5
5.5 EXTRACTION, STORAGE AND TRANSPORT OF SAMPLES .................................... 6
5.6 PREPARATION OF COMPOSITE SAMPLES ......................................................... 7
5.7 LABORATORY TESTING AND ANALYTICAL QUALITY ............................................ 7
5.8 RESEARCH ETHICS ...................................................................................... 8
6. ANNEX 1: LIST OF PERSISTENT ORGANIC POLLUTANTS ...................... 9
7. ANNEX 3: SELECTION GUIDE ................................................................... 12
8. ANNEX 4: DONOR’S CONSENT AGREEMENT ......................................... 13
9. ANNEX 5: LIST OF PARTICIPANTS ......................................................... 14
10. ANNEX 6: QUESTIONNAIRE .................................................................... 15

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1. Introduction
There is general consensus that human breast milk is the best food for infants, especially in
the first six months of life. Not only is breastfeeding important for the infant’s growth and development
because of the high quality and easy digestion of the nutrients in human breast milk, but it is also
important because body contact strengthens mother-and-child bonding and boosts the child’s
psychological and social development. It is based on this knowledge that the Ministry of Health, the
World Health Organization (WHO) and the United Nations Children’s Fund (UNICEF) promote,
encourage, support and protect breastfeeding.
In pursuance of the above, the country coordination must raise awareness on the issue
among all the collaborators involved in the study, highlighting the benefits of breastfeeding for infant’s
health, and stating that the practice should be incentivized. Participants should bear in mind that the
sampling of milk can by no means hinder the feeding and well-being of infants and donors.
The World Health Organization (WHO) and the scientific community in general have
especially focused on the Persistent Organic Pollutants (POPs), owing to their high persistence in the
environment, their high toxicity and potential adverse effects on human health and deleterious effects
on other living beings. In view of the sound evidence of their toxicity, and seeking to promote chemical
safety, WHO has called for international action in the control and reduction of such substances in the
different environmental compartments.
Since 1976, WHO has devoted special attention to these pollutants, and has been collecting
information on POPs in foodstuff, also including human milk. Three international studies were
conducted during the 1987 – 2003 period, to evaluate the levels and trends of POPs in human milk.
Human milk, blood and fatty tissue are relevant matrixes for the evaluation of the body POP
burden, but human milk is recognized as the matrix of choice owing to its many advantages, such as
the easy collection of its samples with a non invasive technique. In addition to providing information on
the exposure of mothers and their children, monitoring of human milk can provide information on the
need for measures to reduce the levels of these substances in foodstuff, the primary source of
exposure in the general population, and consequently in the environment.
The United Nations Environment Program (UNEP) and WHO are currently coordinating
Round 4 of the Study of POPs in Human Milk, in pursuance of the Global Monitoring Program, which is
part of the actions set forth by the Stockholm Convention, in its effort to reduce/eliminate the release of
POPs into the environment.
Below is the list of the people that participated in the development of the document herein:
Dr. Heidelore Fiedler, UNEP, Chemicals
Dr. Ana Maria Cheble Bahia Braga, ENSP/FIOCRUZ
Chem. Thelma Pavesi, ENSP/FIOCRUZ
Dr. Thomas Manfred Krauss, ENSP/FIOCRUZ
Chem. Engineer Alejandra Torre
B.A. Sandra Castro
Chem. Virginia Santana

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2. Documents of reference
Guide for the development of a national protocol, for the Fourth Study on Persistent Organic
Pollutants in Breast Milk, coordinated by WHO in collaboration with the United Nations Environment
Program (UNEP), (Revised on October 1 2007).
3. Objectives
Provide information on the impact of POPs on health by:
Expanding and strengthening the studies of WHO’s Global Environment Monitoring System
(GEMS) on human exposure to all the POPs included in the Stockholm Convention;
Providing data on human exposure to POPs to all the health, environment, agriculture and
fishery sectors, so they can use them when assessing and managing risk;
Identifying needs for future studies, including prospective epidemiological studies.
Provide accessible, reliable and comparable data on the POP levels in breast milk for the
purposes of the Stockholm Convention:
Assist in the development and review of the National Implementation Plan in accordance with
Article 7 of this Convention;
Contribute to the Effectiveness Evaluation of the Stockholm Convention in relation with the
reduction and elimination of POP releases into the environment, as required in Article 16;
Approach relevant measures of Article 11 of the Convention with regard investigation and
monitoring of POPs.
4. Scope
This procedure shall apply to the sampling of breast milk in all the countries that participate in
the GEF Project.

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5. Methodology
5.1 Type, number and volume of samples
This guide only approaches a study with one sampling period. The samples obtained in that
period will be used to determine the reference concentrations of POPs in individual randomly selected
samples of breast milk and in composite samples prepared with them.
The use of composite samples is recommended to determine the concentrations of dioxin-like
PCDD, PCDF and PCB in human breast milk and individual samples to determine basic POP pesticides
and PCB markers. In the document herein, those two groups of contaminants will be referred to as
“analytically simple POPs” and “analytically complex POPs”, respectively. Annex 2 contains a list of the
analytically simple POPs (basic POPs) and complex POPs (dioxin-like POPs) included in this guide, as
well as a list of the breakdown related products, to be tested and reported together with the precursor
compounds.
As an internal analytical quality control method, the analytically simple POPs will also be
tested in the composite sample, because the average value obtained from the individual
samples must be equal to the value in the composite sample. Moreover, composite and/or
individual samples may be used to determine other POPs that are currently not covered by the
Stockholm Convention, based on the country’s priorities and the resources available. Annex 2
provides a list of optional POPs that may be included in the analysis of the composite samples.
In order to obtain statistically reliable data, it is essential to recruit an appropriate number of
individual sample donors. In principle, it is recommended to include a minimum of 50 individual samples
per country. However, it is recognized that countries with a small number of inhabitants, low birth rates,
or both, may require a certain flexibility. If that were the case, the first choice to increase the number of
donors should be to extend the period for collecting samples. Under certain circumstances the
reduction of the number of donors is inevitable; in such cases, investigators must thoroughly weigh the
effect of sample size reduction on the statistical power of the study, to detect differences between
different periods. Conversely, the study’s statistical power may be enhanced by including more than 50
individual samples, and this option is recommended. Especially, countries with over 50 million
inhabitants should include at least one additional participant per million people exceeding 50 million.
Countries whose populations exceed the 50-million threshold by far (or who can afford the resources)
are recommended to prepare a second composite sample (or more) if possible. The Country
Coordinator is responsible for guaranteeing that the number of samples collected for analysis provides
a statistical base, sufficient to ensure that the evaluations of the changes that may occur in the POP
concentrations with time are scientifically valid. Annex 3 contains some statistical aspects to be
considered in the design of the protocol.
5.2 Collection of samples
The collection of breast milk will be coordinated by the national laboratory. Collection should be
performed in breast milk banks (BMBs) when available, or at hospitals, when BMBs are not available in
the country. Below are the advantages of using BMBs for collection of samples:
The sampling is performed by skilled professionals, qualified for collecting breast milk and handling
samples;
Milk Banks have the kind of infrastructure required for an adequate storage of samples;

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The professionals acknowledge the importance of breastfeeding and encourage the potential
donors to breastfeed.
One of the issues to be considered when selecting the Banks is the ease with which the
samples can be sent fast to the laboratory that coordinates the study, located in areas with a high
population density.
All the Banks will be contacted, and after clarifying the purpose of the study, a pre-selection will
be made among those that show interest and willingness to collaborate. The banks selected must
represent their countries. The countries should be divided into more regions and the banks available in
each region should be selected at random, to ensure a vast geographical distribution of the sampling
sites across the country. To determine the total number of Banks needed, the country coordination will
have to:
Determine the total number of individual samples that need to be collected
Consider that at least 5 samples will have to be collected per site, with a maximum of 10 samples
per site;
The same number of samples will be collected per site (UNEP composite sample - 50 persons
- five regions – two BMBs per region – five samples per BMB)
5.3 Training of collaborators
After selecting the breast milk banks and hospitals, the study coordinators will visit the sites
selected to inform about the study details and the training it involves, the selection of donors and the
implementation of the questionnaire, as well as to clarify any doubts that may arise when the
procedures are implemented. At that briefing visit, the coordinators will leave the previously prepared
collection jars and all the material that will be needed (e.g.: questionnaires).
5.4 Selection of donors and implementation of questionnaire
The potential donors will be recruited among mothers that seek a milk bank to donate milk or
who donate milk at the hospital. Primarily, they must meet the selection criteria recommended in the
guide developed by WHO:
Primiparas;
Mothers not older than 30 years of age;
Both mother and child must be apparently healthy, and pregnancy had to be normal;
The mother must be breastfeeding only one child (e.g.: no twins);

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Only mothers that have lived at least ten years in the area must be included;
Only mothers that are exclusively or primarily breastfeeding must be included;
The mother must not live in the vicinity of incinerators, paper mills, metal factories and chemicals
factories;
The mother must be available for the extraction of the sample between the third and eighth week
postpartum.
The mothers that meet all these requirements will be invited to participate in the study by signing the Consent
Agreement (Annex 4). Each mother included in the study must be identified with a single code, the same that
will appear by her name in the List of Participants (Annex 5). This code will be recorded in the Selection Guide,
in the Consent Agreement, in the Individual Questionnaire and the collection jar. Before the milk is collected,
the donor will be interviewed by a milk bank professional, and will provide information about her housing and
feeding habits, in accordance with the Individual Questionnaire (Annex 6).
5.5 Extraction, storage and transport of samples
The collection will be conducted at the human milk banks between the 3 th and 8 th weeks postpartum. The
total volume of the breast milk to be sampled from each mother will be 100 ml. This volume can be obtained
easily without impairing the infant’s feeding.
No other containers may be used to collect the milk. Preferably, the extraction must be performed manually,
expressing the milk directly into the jar provided for that purpose.
Breasts and hands should be maintained as clean as possible, avoiding the use of soap. When the mother is
using some ointment (for cracked nipples, or other medical reasons), the application must be limited to the
moments between the extractions of milk, and the product should be completely removed, washing the breasts
prior to the procedure.
Each mother must be given an identification code number, to match the code on the milk collection jar. The
jars are supplied by the study coordinators, after they have undergone an adequate chemical treatment to
ensure they are free from contamination, and with three potassium dichromate pellets to preserve the samples
appropriately. The sample must be collected directly into the container; if the sample is taken at the mother’s
home, the jar must be refrigerated until its delivery. The sample may be kept in the refrigerator, at
approximately 4 °C, for no longer than 72 hours, or in the freezer, at -20 °C, for longer periods. If the sample
cannot be refrigerated, you can add a small tablet of potassium dichromate (K2Cr2O7) for chemically sterilized
milk.
After the collection of samples, the fifty 50-ml samples must be sent to the laboratory appointed by the
Country Coordinator. Sample shipment must be performed by professional carriers or through other means, as
expeditiously as possible. Samples are frozen at -20 °C, wrapped in dry ice and sent to destination. In the
countries where the temperature cannot be controlled, the samples are preserved by adding 100 mg of
potassium dichromate every 250 ml of milk. All the individual or composite samples must carry a label with one
single identification code. The composite samples must be sent to the WHO Reference Laboratory, together
with the summarized information (see Annex 8). The laboratory that receives the samples must be informed on
the shipping date and the expected date of arrival, and must confirm their reception.

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5.6 Preparation of composite samples
The samples must be handled by qualified personnel, to guarantee their integrity. Individual milk
samples must be homogenized by heating them at 38 °C and shaking them for 10 minutes, after which
the laboratory must prepare individual samples and composite samples. The individual samples must
contain 25 ml of breast milk for the analysis of analytically simple POPs, as pesticide POPs and PCB
markers. As to the composite sample, 10 ml of each one of the 50 individual samples must be taken to
obtain a 500-ml composite sample. Of those 500 ml, 50 ml must be kept aside for the analysis of simple
POPs by the participant countries, and the rest, 450 ml, will be analyzed by the WHO Reference
Laboratory. The latter will analyze both analytically simple and complex POPs, such as dioxin-like PCBs,
PCDD and PCDF. The rest of the sample collected from individual donors, (15 ml), must be mixed to
obtain a 750-ml composite sample that will be sent to WHO for its Human Milk Global Bank. The Bank’s
reserve will be used in the future if new POPs are added to the Stockholm Convention, as well as for
other scientific purposes. Countries with adequate resources may opt to obtain more than one
composite sample from the 50 individual samples; for example, one composite sample obtained with 25
individual samples. However, not all the composite samples need to contain 500 ml. If the country
wishes to analyze other contaminants in aliquots of the composite samples, the protocol for sample
collection will need to be modified accordingly (either to include more donors or to increase the volume
of each milk sample). More information is available at the WHO Reference Laboratory.
5.7 Laboratory testing and analytical quality
All the composite samples will be tested at the Laboratory for Dioxins and Agrotoxic Substances
of the "Chemisches and Veterinäruntersuchungsamt Freiburg" (CVUA: Community Reference
Laboratory for Chemical Testing of Foods of Animal Origin), in Freiburg, Germany. This laboratory was
appointed as a WHO Reference Laboratory for the study, having met all the requirements established by
WHO at the Fourth Round for the Interlaboratory Calibration of PCDD, PCDF and PCB levels in human
milk.
The Reference Laboratory follows a rigorous quality control program to guarantee the accuracy
and reliability of the results obtained in the study. The laboratory runs this program daily in the routine
testing for PCDD, PCDF and PCB and POP agrotoxics, primarily on foodstuff samples.
The elements below are part of the quality program:
Testing of reagent blank samples;
Testing of fortified samples (refined vegetal oil);
Testing of reference samples (known concentrations);
The analytical procedures for the PCDD/PCDF and PCB will be performed in accordance with
the procedure described by Malisch and Leeuwen (2002) 1 . Following freeze-drying, the sample will be
extracted with ethanol/toluene (70:30, v:v) for 8 hours in a "Twisselmann" extractor. The raw extract,
primarily consisting of fat, will be purified with ether and fortified with PCDD/PCDF and PCB in-house
standards labeled with 13 C. The fat from the extract will be removed using gel permeation
chromatography (Bio Beads S-X3). After treating the extract in a multilayer column (silica, silica/sulfuric
acid), the PCDD/PCDF and PCB will be separated in a Florisil column. The PCDD/PCDF fraction will be
treated in an activated charcoal column and later analyzed in a High Resolution Gas Chromatographer
1 Malisch, R; Leeuwen FXR. (2002). Organohalogen Compounds 56, 317-320.

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coupled to a Low Resolution Mass Spectrometer (HRGC/LRMS). The PCB fraction will also be treated
in an activated charcoal column to separate it into three fractions: non-ortho, mono-ortho and di-ortho
PCB. Each fraction will be analyzed with HRGC/LRMS.
The analytical method for agrotoxic POPs will be based on the principles set forth in the Official
Methods of the German Federal Food Act, for the Analysis of Agrotoxic Residues in Foodstuffs 2 .
Following freeze-drying, the sample will be extracted with ethanol/toluene (70:30, v:v) for 8 hours in a
"Twisselmann" extractor. The raw extract, primarily consisting of fat, will be purified with ether and
fortified with in-house standards. The fat will be removed from the extract through gel permeation
chromatography (Bio Beads S-X3). The extract will subsequently treated in a partially deactivated silica
column. The determination of the agrotoxic POPs will be performed in a gas chromatographer with
electron capture detector (ECD). The results will be confirmed in a High Resolution Gas
Chromatographer coupled to a Low Resolution Mass Spectrometer.
The analysis of agrotoxic POPs and the PCB markers in the individual samples will be performed
at the national POP Laboratory, if available.
5.8 Research Ethics
The human milk samples collected and the questionnaires completed will be used specifically
for the purposes of this research, and shall not be employed for other purposes. Any data provided by
mothers that may permit their personal identification, will be maintained confidential and shall not be
disclosed.
2 Collection of Official Methods under Article 35 of the German Federal Food Act, Modular Multiple Analytical
Method for the Determination of Pesticide Residues in Foodstuffs, L 00.00-34, November 1999

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6. Annex 1: List of Persistent Organic Pollutants
A. 12 Initial POPs
Simple analysis POPs - Agrotoxic POPs and PCB markers
Aldrin
Chlordane (total)
alpha-chlordane (α-chlordane)
gamma-chlordane (γ-chlordane)
oxi-chlordane
cis-nonachlor
trans-nonachlor
Heptachlor (total)
Heptachlor
Heptachlor epoxide (cis)
Heptachlor expoxide (trans)
Hexachlorobenzene (HCB)
Dieldrin
Mirex
DDT (total)
Toxaphene (total)
o,p'-DDD and p,p'-DDD
o,p'-DDE and p,p'-DDE Parlar 26
o,p'-DDT and p,p'-DDT Parlar 50
Parlar 62
Endrin (total)
Endrin
Endrin Ketone
PBC Markers
IUPAC # 28
IUPAC # 52
IUPAC # 101
IUPAC # 118
IUPAC # 138
IUPAC # 153
IUPAC # 180

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Dioxin-Like POPs
Polychlorinated Dibenzo-p-dioxins and Polychlorinated Dibenzofuranes
Polychlorodibenzo-p-dioxins (PCDD)
2,3,7,8-Cl4DD
1,2,3,7,8-Cl4DD
1,2,3,4,7,8-Cl6DD
1,2,3,6,7,8-Cl6DD
1,2,3,7,8,9-Cl6DD
1,2,3,4,6,7,8-Cl7DD
Cl8DD
Polychlorodibenzofuranes (PCDF)
2,3,7,8-Cl4DF
1,2,3,7,8-Cl5DF
2,3,4,7,8-Cl5DF
1,2,3,4,7,8-Cl6DF
1,2,3,6,7,8-Cl6DF
1,2,3,7,8,9-Cl6DF
2,3,4,6,7,8-Cl6DF
1,2,3,4,6,7,8-Cl7DF
1,2,3,4,7,8,9-Cl7DF
Cl8DF
Dioxin-like PCBs
non-ortho PCB
mono-ortho PCB
3,3',4,4'-TeCB (PCB 77) 2,3,3',4,4'-PeCB (PCB 105)
3,4,4',5-TeCB (PCB 81) 2,3,4,4',5-PeCB (PCB 114)
3,3',4,4',5-PeCB (PCB 126) 2,3',4,4',5-PeCB (PCB 118)
3,3',4,4',5,5'-HxCB (PCB 169) 2',3,4,4',5-PeCB (PCB 123)
2,3,3',4,4',5-HxCB (PCB 156)
2,3,3',4,4',4'-HxCB (PCB 157)
2,3',4,4',5,5'-HxCB (PCB 167)
2,3,3',4,4',5,5'-HpCB (PCB 189

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Nine new POPs (Listed by the Fourth Conference of the Parties, May 2009)
Chlordecone
Hexachlorocyclohexane (HCH) (total)
Chlordecone
alpha-hexachlorocyclohexane (α-HCH)
beta-hexachlorocyclohexane (β-HCH)
gamma-hexachlorocyclohexane (y-HCH)
Pentachlorobenzene
Polybrominated diphenyl ethers (PBDE)
BDE 47
BDE 99
BDE 153
BDE 154
BDE 175/BDE 183
Hexabrominated biphenils (HxBB)
PBB 153
PFOS/PFOA
PFOS - (anion)

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7. Annex 3: Selection Guide
CONFIDENTIAL
Selection Guide
For donors of breast milk for the testing of Persistent Organic Pollutants (POPs)
Place of interview: _________________________________________ Date of interview:___/___/___
Interviewee N°:____
Selection criteria:
1. Child more than 2 weeks old and less than 8 weeks old
2. First child
3. Mother not older than 30 years of age
4. Normal pregnancy
5. Mother and child apparently healthy
6. Mother breastfeeds only one child
7. Exclusive breastfeeding, preferably
8. Mother has lived in the area for 10 years
9. Mother does not live near an incinerator, a paper mill, a metal factory or
a chemicals factory
Yes
No
If all the answers are affirmative, invite the mother to participate in the study and ask her to sign her consent so
it can be included in the study.
Mother accepted to participate
Mother signed the consent agreement
Yes
No
If the mother signed the consent agreement, she is to be included in the study; record her name and address in the
List of Participants and determine her appropriate code. Record this code in all the documents and use the jar with
the same code for the milk sample.
Identification code of the sample: ______________________
Interviewer (Full name in bold types): ____________________________________
Signature:_________________________________________________________

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8. Annex 4: DONOR’S CONSENT AGREEMENT
I, ________________________________________________________________________________
freely accept to participate in the study "Levels of Persistent Organic Pollutants in Breast Milk”,
conducted by…………………………. and the World Health Organization/United Nations Environment
Program, under the coordination of the investigators……………….. (e-mail:………………….) and (email:………………………….),
address:………………………. , who may be contacted any time during
the research.
Purpose of the study: The study is aimed at quantifying the levels of Persistent Organic Pollutants in
human milk to estimate the levels of human exposure to POPs and the environmental pollution caused
by these agents. POPs bio-accumulate in animal fat; as human breast milk is rich in fat, the dosing of
these substances reflects the degree of exposure of the general population, without requiring invasive
and painful extractions.
Study Rationale: The study gives a general view of the population’s levels of exposure in several
geographical areas in the country. This information and the knowledge developed enables government
agencies to adopt measures to control the environmental pollution caused by these contaminants and
to reduce the health hazards for the general population.
Participation: Once my participation is accepted, 100 ml of breast milk will be extracted in private. In
addition, I will answer a questionnaire on my personal habits, data on pregnancy, medicines, my work
and my feeding habits. As in any study, unforeseeable risks may arise, but all the precautions will be
taken to protect my personal safety during the phases of the study herein.
Benefits: The information obtained in this study can be of scientific use and it may be useful for others.
In addition, the presence of these agents in question at hazardous levels in breast milk will be included
in the donor mother’s data, attaching due health care measures, and provisions aimed at reducing
environmental pollution.
Privacy: Any personal information obtained in the investigation herein shall be confidential and shall
only be disclosed with my authorization. The data referring to me shall be given to the investigators in
charge. The scientific data and the medical information derived from the study herein can be presented
at conferences and published in journals without my identification.
I hereby state that my participation in this study is voluntary and that I am aware that my decision not to
participate or to quit the study after being enrolled will not entail any problems to the
………………………………………….., WHO, UNEP or any other agency or institution involved. I can
terminate my participation in the study at any time and for any reason, needing no justifications.
Signature of donor mother
Signature of witness
Date: _____ / ____ /
Investigator
Identification code:________________________________

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9. Annex 5: List of Participants
Study of the Levels of Persistent Organic Pollutants in Human Milk
Place of Extraction: XXXXXXX/YY Confidential !
Sample code: Name of donor mother Address of donor mother Telephone
ZZ-YY-01
ZZ - YY -02
ZZ - YY -03
ZZ - YY -04
ZZ - YY -05
ZZ - YY -06
ZZ - YY -07
ZZ - YY -08
ZZ - YY -09
ZZ - YY -10

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10. Annex 6: Questionnaire
For donors of breast milk for the analysis of Persistent Organic Pollutants
CONFIDENTIAL!
Questionnaire
1. Country: 2. Area (city or region within the country): 3. Identification code of composite
sample:
XXXXXXX
ZZ-XX
4. Sample identification code: 5. Start and end of extraction
6. Date of mother’s birth:
ZZ-XX___
From: DD/MM/YY
To: DD/MM/YY
DD/MM/YY
7. Mother’s age in years: 8. Mother’s height in cm: 9. Mother’s weight before
pregnancy in kg
10. Mother’s current
weight in kg:
11. Area of residence in the last 10 years:
Urban
Rural
12. Previous area of residence:
Years
urban
rural
And earlier
Years
urban
rural
13. Child’s age in
weeks at start of
sampling:
14. Child’s sex:
Boy
15. Child’s birthweight
in grams:
16. Child’s weight at sampling
in grams
First day:
Girl
en
Last day:

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17. Mother’s feeding habits before pregnancy:
Varied diet Vegetarian, but includes milk and eggs Only vegetarian
Others
Description: _________________________________________
18. Has the mother changed her feeding habits significantly Yes No
since she got pregnant
If yes, describe how:___________________________________________________________
___________________________________________________________________________________
19. How often (as an average) did the mother consume fish during pregnancy
Never Less than once a week Once a week
Twice a week More than twice a week Every day
Describe the species more frequently consumed by the mother:
___________________________________________________________________________________
____________________________________________________________________________________
20. How often (as an average) did the mother consume sea food during pregnancy
Never Less than once a week Once a week
Twice a week More than twice a week Every day
Describe the species more frequently consumed by the mother:
_______________________________________________________________________________
_______________________________________________________________________________

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21. How often (as an average) did the mother consume sea mammals (e.g. whale) before pregnancy
Never Less than once a week Once a week
Twice a week More than twice a week Every day
Describe the species more frequently consumed by the mother:
_______________________________________________________________________
_______________________________________________________________________
22. How often (as an average) did the mother consume milk before pregnancy
Never Less than once a week Once a week
Twice a week More than twice a week Every day
Type of milk:_______________________________________________________________
Daily consumption:
Less than 250 ml 250 – 499 ml 500 ml or more
23. How often (as an average) did the mother consume cheese before pregnancy
Never Less than once a week Once a week
Twice a week More than twice a week Every day
Type of cheese: __________________________________________________________________
24. How often (as an average) did the mother consume other dairy products (e.g. butter, yoghurt) before
pregnancy
Never Less than once a week Once a week
Twice a week More than twice a week Every day
Type of product: ________________________________________________________________

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25. How often (as an average) did the mother consume meat before pregnancy
Never Less than once a week Once a week
Twice a week More than twice a week Every day
Describe the type of meat more frequently consumed by the mother:
_______________________________________________________________________
_______________________________________________________________________
26. How often (as an average) did the mother consume eggs before pregnancy
Never Less than once a week Once a week
Twice a week More than twice a week Every day
27. Mother’s smoking habits:
She does not smoke Former smoker Current smoker
If she smokes, what does she smoke Cigarette Cigar Pipe
If she smokes cigarettes, how many a day
If she smokes cigars, how many a day
28. Did the mother work before pregnancy
Yes
No
If the answer is yes, describe the type of job (function, activities, contact with hazardous goods, etc.)
and duration in years:
____________________________________________________________________________
_____________________________________________________________________________

Standard Operating Procedure
for POP Sampling in Breast Milk
Page 19 of 20
29: Types of past jobs:
30. Duration in years
a)___________________________________________________________
___________________________________________________________
b)__________________________________________________________
__________________________________________________________
Never worked
31. Was the donor’s mother born in Brazil Yes No
31. Was the donor herself breastfed
Yes No I don’t know
If the answer is yes, how long_________________
32. Was the donor’s house treated with DDT to kill mosquitoes
Yes No I don’t know
If the answer is yes, when_________________
33. Medicines used during the sampling period, for how long and at what dosages (including vitamins and natural
supplements):
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
Questionnaire completed by:
(bold types)
Date:
Signature: